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NATIONAL INSTITUTES OF HEALTH 



Review of 

INTRAMURAL RESEARCH 



1960 




U.S. DEPARTMENT OF 

HEALTH, EDUCATION, AND WELFARE 

Public Health Service 



brary 

ifonal Institutes of Health 

lllfeli Maryland 



/ 



ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 



NATIONAL INSTITUTES OF HEALTH 



Review of 
INTRAMURAL RESEARCH 




U. S. DEPARTMENT OF HEALTH, WELFARE, AND EDUCATION 



PUBLIC HEALTH SERVICE 



NATIONAL INSTITUTES OF HEALTH 




\. use of 

: a 



INSTITUTE RESEARCH DIRECTORS 



National Cancer Institute Charles G. Zubrod, M.D. 

Scientific Director 

Nathaniel I. Berlin, M.D. 

Clinical Director 

National Heart Institute Robert W. Berliner, M.D. 

/Scientific Director 

Donald S. Fredrickson, M.D. 

Clinical Director 
National Institute of Arthritis 

and Metabolic Diseases De Witt Stetten, M.D. 

Scientific Director 

Joseph J. Bunim, M.D. 

Clinical Director 
National Institute of Allergy 

and Infectious Diseases Dorland J. Davis, M.D. 

Scientific Director 

Vernon Knight, M.D. 

Clinical Director 

National Institute of Mental Health John C. Eberhart, M.D. 

Scientific Director 

Robert A. Cohen, M.D. 

Clinical Director 
National Institute of Neurological 

Diseases and Blindness G. Milton Shy, M.D. 

Scientific Director 

Maitland Baldwin, M.D. 

Clinical Director 

National Institue of Dental Research . . . Seymour Kreshover, M.D., D.D.S. 

Scientific Director 

Robert M. Stephan, D.D.S. 

Clinical Director 

Division of Biologics Standards Roderick Murray, M.D. 

Scientific Director 



G. Burroughs Mider, M.D. 
Director of Laboratories and Clinics 

Thomas J. Kennedy, M.D. 
Assistant to Director of Laboratories and Clinics 



m 



FOREWORD 



The National Institutes of Health, one of the five Bureaus of the United States Public 
Health Service, has been assigned the mission to conduct and to support research, re- 
search training, and other related activities. This mission is discharged partially through 
an extramural support program which, administered from Bethesda, reaches into vir- 
tually every institution in the United States engaged in biomedical research, and is 
rapidly expanding throughout the world. And it is partially discharged through a 
direct operation, the intramural program, housed in laboratories in Bethesda, and repre- 
senting something of a microcosm of the total effort. This publication focuses exclusively 
on our intramural enterprise, describing it in a series of reviews designed to illustrate 
the compass and flavor of the local research activities of each of our Institutes. Thus, 
it is not a comprehensive presentation of the program of either the United States Public 
Health Service or of the National Institutes of Health. 

Glimpses of the extent to which the intramural research effort has participated in 
the broad forward thrust of medical research are provided in the eight Annual Sum- 
mary Reports comprising this compendium. The reports are protected from editorial 
interference, and prepared by the originators in accord with only the most general guide- 
lines. This procedure, considered and deliberate, has been adopted to allow the reader 
to savor the diversity of outlook and attitude which prevails among a group of brilliant 
scientists loosely knit and almost imperceptibly harnessed for the attainment of common 
categorical goals. In such a presentation, it is possible to overlook the thread of mission 
that ties each of these operating research organizations into the broad program of the 
National Institutes of Health. It is appropriate, therefore, to pursue this aspect briefly. 

The mission of the National Institutes of Health as a whole and of its components 
is to develop the facilities, resources, and attitudes which would be most effective in 
acquiring new knowledge concerning disease processes, to the end of relieving suffering, 
bringing about cure and rehabilitation, and assuring the prevention, whenever possible, 
of disease. Broadly considered, this mission involves providing the wherewithal and 
cultivating suitable soil for a systematic study of man and his milieu with the ultimate 
objective of contributing to improved health. From this overall point of view, the NIH 
does not differentiate between what is done intramurally and extramurally. However, 
within these conceptions are contained more specific objectives only some of which can 
be sought for within an intramural research program, while the others may be searched 
out most expeditiously by support of work in other institutions through the extramural 
program. 

This second Annual Review of NIH Intramural Research provides evidence of the 
magnitude of the effort — both in breadth and depth — and of the type of achievements 
that have placed this installation in the forefront of research in the medical sciences. 



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James A. Shannon, 

Director. 



CONTENTS 



Page 

foreword • "*" 

National Cancer Institute 1 

Introduction 1 

Clinical investigations 3 

Origins of the cancerous state 4 

Development of clinical cancer 5 

Autonomy 5 

Spread of cancer 6 

Wound washings 6 

Cancer cells in circulating blood 7 

Spread of tumor in animal models. . 7 

Effects of cancer upon the patient 7 

Direct effects of cancer growth , 8 

Blood-forming tissues — platelets 8 

Leucocytes and immunity 8 

Gastrointestinal tract 9 

Bone metastases 9 

Indirect effects of cancer growth 9 

Hormonal products of cancer 9 

Indirect effects on erythrocytes 10 

Unexplained indirect effects of cancer 10 

Management of cancer 11 

Surgery 11 

Operative effectiveness 11 

Complications of radical surgery 12 

Wound infections 12 

Operative and postoperative blood loss 12 

Radiation IS 

Clinical studies 13 

Animal studies 14 

Chemotherapy ','. 15 

General remarks 15 

The intramural chemotherapy pro- 
gram of NCI 15 

Specific antitumor agents 15 

Conclusion 19 

Nonclinical investigations 20 

Studies on the induction of cancer 20 

Chemicals 21 

Mechanisms of carcinogenesis 23 

Viruses 25 

Virologlcal aspects of human tumors. 25 

i , Moloney virus 26 

Polyoma virus 28 

Mammary tumor agent 30 

Rous sarcoma virus 30 

, Other virus studies 31 

Host — tumor relationships 33 

Host genetics in carcinogenesis 33 

Radiation studies 34 

Radiation of cells in tissue culture . . 35 



Page 

Immunological phenomena and polysac- 
charide studies 35 

Endotoxin studies 36 

Tumor cytotoxic factors in serum ... 37 
Antigens against human carcinomas. 37 
Immune tolerance induced by breed- 
ing 38 

Polysaccharide investigations 38 

Metabolism of the tumor-bearing host ... 40 

Body composition studies 40 

Chemically defined diets 41 

In vivo perfusion studies 42 

Kinetic studies in tumor-bearing ani- 
mals 43 

Enzyme activities of the tumor-bear- 
ing host 43 

Deoxyribosldes of the tumor-bearing 

host 44 

Urinary excretion patterns of nucleic 

acid congeners in leukemia patients. 44 

Vector- Analysis Computer Techniques . 45 

Energy expenditure studies 45 

Tumors and their properties 45 

Plasma cell tumors 47 

Diffusion chamber studies 48 

Tissue culture studies 49 

Transformation studies 50 

Immunological aspects , . . 51 

Enzymology 52 

Studies on proteins and nucleic acids ... 58 

Protein characterization 52 

Protein synthesis studies 53 

Amino acid and peptide synthesis ... 55 

Nucleic Acids 55 

Enzymatic studies 57 

Chemotherapy investigations 59 

Tumor assay systems 60 

The advanced leukemia L1210 assay 

system 60 

The sarcoma 37 assay system 61 

The adenocarcinoma 755 assay system 61 

Ehrlich ascites tumor 61 

Hepatoma 129 61 

Moloney virus leukemia 62 

Spontaneous tumors 62 

Biochemical studies 62 

Drug resistance studies 63 

Glycolysis studies 64 

Chemotherapy of experimental central 

nervous system leukemia 65 

Service functions 65 



VIII 



CONTENTS 



Page 

National Heart Institute 67 

Introduction 67 

Laboratory of cellular physiology and metabolism 67 

Section on cellular physiology 67 

Section on metabolism 70 

Metabolic activity of adipose tissue 70 

Triglyceride synthesis 70 

Influence of hormones 70 

Physiology of adipose tissue and the 

mechanism of free fatty-acid release . . 71 

Biosynthesis of cholesterol 71 

Hypoproteinemia and hyperlipemia of ne- 
phrosis 71 

Relation of serum FFA levels to lipid 

deposition in tissue 72 

Clinical studies of hypoproteinemia 72 

Fibrinogen 72 

Fatty-acid transport 72 

Regulation of hipoprotein synthesis 73 

Cholesterol catabolism 73 

Rate of lipoprotein synthesis in vitro 73 

Epinephrine — induced hyperlipidemia 73 

Defective lipid metabolism 73 

Plasma post-heparin lipolytic activity 73 

A new lipodosis 74 

Section on enzymes 74 

Lipid metabolism 74 

Fatty-acid synthesis in C. klyyveri... 74 

Fatty-acid synthesis by adopise tissue 74 

Propionate metabolism 75 

Metabolism of onium compounds 75 

Anaerobic fermentation of choline . . 75 

Sulfonium compounds 76 

Metabolism of heterocyclic compounds ... 76 

Riboflavin degradation 76 

Biosynthesis of phenazine-1-carboxylic 

acid 76 

Regulation of biosynthetic pathways 77 

Aspartate metabolism 77 

Metabolism of amino acids 77 

Conversion of phosphohomoserine to 

threonine 77 

Sulfur transfer between homocysteine 

and cysteine 77 

Gamma-aminobutyric acid metabolism 77 

Reduction Deamination of glycine .... 78 

One-carbon metabolism 78 

Lipoate in metabolism of lactate 78 

Pyruvic kinase action 79 

Biochemistry of the differentiating slime 

mold 79 

Carbohydrate metabolism 79 

Isocitrate and glucose-6-p-dehydrogen- 

ases 79 

Enzyme induction 79 

Laboratory of chemistry of natural products 79 

Lipid methodology 80 

Alkaloid work 80 

Kallikrein-kallidinogen-kallidin system 81 

Laboratory of chemical pharmacology 81 



Page 

Development of New Drugs 81 

Reserpine analogues 81 

Benzoquinolizines with reserpine-like ac- 
tion 81 

Imipramine metabolite 81 

Drugs for arthritis and gout 81 

Problems of drug administration in long-term 

therapy 82 

Biochemically irreversible drugs 82 

Effects of drugs on endocrines 82 

Drug combinations 82 

Biogenic amines 82 

Serotonin (5HT) and norepinephrine 

(NE) in brain 82 

Brain amines in the newborn 83 

Imipramine (tofranil) 83 

Monoamine oxidase inhibitors 83 

Hypotensive drugs — guanethidine and bre- 

tylium 83 

NE in sympathetic synaptic transmission 84 

Reserpine action 84 

Histamine 84 

Studies in biochemical behavior 84 

Passage of substance across membranes 85 

Membranes within the CNS 85 

Penetration of drugs into cells 85 

Active transport mechanisms 85 

Transport of catecholamines 85 

Effect of ouabain on phosphatide acid. .'. : 86 

Drug metabolism 86 

Antimetabolites 86 

Microsomal drug oxidation 86 

Induced enzyme formation 86 

Studies with ascorbic acid 86 

Chemical inhibition of cholesterol synthesis. . 87 

Development of methods of analysis 87 

Laboratory of technical development 87 

Laboratory of cardiovascular physiology 89 

Homeometric autoregulation in the heart 89 

The atrium 89 

Mitral valve closure 89 

Left atrial and left ventricular end dias- 
tolic pressure 90 

Catecholamine metabolism of the heart. . 90 

Peripheral circulation 90 

Carotid sinus activity and oxygen con- 
sumption 90 

The kallikrein system 90 

Reflex factors in renal vascular resistance. . . 91 

Pulsus alternans 91 

Chemoreceptors 91 

The carotid body 91 

Rise in arterial pressure following occlu- 
sion _ 92 

Mammalian myocardium 92 

Shortening and tension development in 

heart muscle 92 

Laboratory of kidney and electrolyte metabolism. 93 

Laboratory of clinical biochemistry 97 



CONTENTS 



IX 



Page 

Laboratory of clinical biochemistry — Continued 

Amine biogenesis and metabolism 97 

Choline biogenesis 99 

Aminobutyric acid metabolism 99 

Amino acid transport 99 

Mechanisms of aromatic hydroxylation 100 

Collagen and hydroxyproline 100 

General medicine and experimental therapeutics 

branch 100 

Section on clinical endocrinology 100 

Section on experimental therapeutics 102 

Biogenic amines 102 

Amino acid metabolism 103 

Action and metabolism of drugs 103 

Section on cardiodynamics 104 

Instrumentation 104 

Cardiovascular biophysics 105 

Pulmonary biophysics 105 

Surgery branch 106 

Gerontology branch 110 

Physiological studies 110 

Longitudinal studies 110 

Renal studies Ill 

Body composition studies Ill 

Energetics of arm motion Ill 

Psychological studies Ill 

Basic biology 112 

Cellular and comparative physiology 112 

Nutritional biochemistry 113 

Intermediary metabolism 115 

Biophysics 116 

Molecular biology 117 

Structure of nucleic acids 117 

Structure of hemoproteins 117 

Metal ions in enzymatic reactions 118 

National Institute of Allekgy and Infectious 

Diseases 119 

Introduction 119 

Laboratory of clinical investigation 121 

Infection of volunteers with respiratory 

viruses 121 

New anti-fungal drug 121 

Simian malaria 121 

Penicillins and penicillinase 122 

Ascites in mice by injection of adjuvants .... 122 

Hypogammaglobulinemia 122 

Bentonite flocculation test 122 

Cystic fibrosis of the pancreas 123 

Rocky Mountain laboratory , . . 123 

Hypersensitivity 123 

Pollovirus 123 

Endotoxins in bacterial fractions 124 

Tuberculosis 124 

Q fever 124 

Other rickettsioses 125 

Bacterial vaccines 125 

Arbor viruses 125 

Colorado tick fever . 126 



Page 

Laboratory of tropical virology 126 

Virus isolates 126 

Eastern equine encephalomyelitis 126 

Encephalomyocarditis 127 

Enterovirus flora in Central America 127 

Mycotic diseases in Panama 127 

Arbor virus 127 

Laboratory of bacterial diseases 128 

Intracellular parasitism 128 

Brucellosis 128 

Laboratory of cell biology 128 

Metabolism of normal cultured cells 128 

Viral synthesis 129 

Galactosemia cell cultures i29 

Laboratory of germ-free animal research 129 

Animal studies 129 

Guinea pigs 130 

Mouse colony 130 

Tumors 130 

Laboratory of parasite chemotherapy 130 

Malaria — human 131 

Malaria — simian 131 

Field studies in Malaya 132 

EE stages and drug action 132 

Insect tissue culture 132 

Biochemical studies 132 

Intestinal parasites 132 

Schistosomiasis 132 

Laboratory of parasitic diseases 133 

Toxoplasmosis 133 

Amoebiasis 133 

Parasitic infections in germ-free animals .... 134 

Sterile culture of worms 134 

Nutrition and schistosomiasis 134 

Dual virus and helminth infections 134 

Ammonia toxicity in mice 135 

Helminths 135 

Laboratory of biology of viruses 135 

Intracellular location of poliovirus 135 

Mutants of EMC virus 136 

Polyoma virus 136 

Tetracycline fluorescence 136 

TMV model 136 

Laboratory of immunology 136 

Allergic thyroiditis 136 

House dust allergens 137 

Genetics of gamma globulin 137 

Hypersensitivity 137 

Human serum auto-antibodies 137 

Fluorescent antibody staining of malarial 

parasites 138 

Laboratory of infectious diseases 138 

Virus pneumonia 138 

Primary atypical pneumonia 139 

Common colds and viruses 139 

New serological test procedures 139 

Serologic reagents 140 

Reference laboratory for viruses 140 

Cancer viruses 140 



X 



CONTENTS 



Page 

Laboratory of infectious diseases — Continued 

Mouse polyoma cancer virus 141 

Extraneous cancer viruses 141 

Medical mycology 142 

Cryptococcus neoformans 142 

Emmonsia crescens 142 

Staphylococcus studies 142 

Streptococcal M protein 143 

Bacterial metabolism 143 

National Institute of Akthkitis and Metabolic 

Diseases 145 

Basic research 145 

Introduction 145 

Administration 145 

Research and education 145 

Laboratory of molecular biology 146 

Organizational matters 146 

Laboratory of nutrition and endocrinology . . . 147 

Nutrition 147 

Vitamin E and factor 3 147 

Folic acid 149 

Vitamin B^ 150 

Germ-free studies 150 

Protein 150 

Lipids 151 

Glucose tolerance factors 152 

Obesity 152 

Guinea pigs 152 

Rabbits 152 

Endocrinology 153 

Experimental diabetes 153 

Pituitary hormones 154 

Steroids 154 

Laboratory of biochemistry and metabolism. . 155 

Carbohydrate metabolism 155 

Biosynthesis of GDP-L-fucose 156 

Nucleic acids 156 

Steroids 157 

Aldehyde dehydrogenase 157 

Metabolism of steroids 157 

Regulatory mechanisms and hormones . . . 158 

Pyridine nucleotides and other coenzymes 158 

Protein and amino acid synthesis 159 

Histidine biosynthesis 159 

Enzymatic utilization of model compounds 159 

Laboratory of physical biology 159 

Laboratory of chemistry 164 

Medicinal chemistry 164 

Carbohydrates 165 

Metabolites 165 

Steroids 168 

Analytical services 169 

Laboratory of pathology and histochemistry.. 169 

Altitude effects 169 

Cytogenetic studies 169 

Degenerative joint disease and human 

rheumatism 170 

Germ-free animals — nutritional deficien- 
cies 170 



Page 

Goldthioglucose obesity 170 

Hemoglobin 170 

Hematology 171 

Histochemistry 171 

Human pathology 172 

Immunology 172 

Renal structure and function 173 

Laboratory of pharmacology and toxicology. 173 

Spermidine and spermine 173 

Histidine and related compounds 173 

Sialic acid 173 

Burns and shock 174 

Mouse leprosy 174 

Sulfur amino acids 174 

Enzyme activity 174 

Excitable cells 174 

Gramicidin J 174 

Cholesterol synthesis 174 

Office of mathematical research 174 

Clinical investigations 176 

Arthritis and rheumatism branch 176 

Serological factors in connective tissue 

diseases 176 

Action of steroids on enzyme systems . . . 177 
Inhibition of DPNH-Cytochrome C 

reductase 177 

Decarboxylation of pyruvic acid .... 177 
Inhibition of glutamic dehydrogenase 

(GDH) reaction 178 

Purine metabolism in gout 180 

Aromatic amino acids 180 

Ascorbic acid in tyrosine metabolism 180 
New toxic effect of corticosteroid therapy 

(posterior subcapsular cataract) .. 181 

New antirheumatic drugs 181 

6-Alpha fluorotriamcinolone 181 

Hydroxychloroquine 181 

An anti-metabolic agent 182 

Gastroenterology 182 

Effects of radiation and folic-acid anti- 
metabolites on intestinal absorption ... 182 
Malabsorption and osteoporosis .......... 182 

Juvenile and adult celiac disease 182 

Protein metabolism in malabsorption 

states 183 

Mammalian metabolism of bile pigments. 183 

Clinical endocrinology branch 183 

Carbohydrate metabolism 183 

Glucose 183 

Insulin 184 

Galactose 185 

Biochemistry of the thyroid 185 

Iodide transport 185 

Phospholipid metabolism 186 

Iodoproteins 186 

Thyroxine and iodotyrosine synthesis. 187 

Hormone transport in blood 187 

Effect of thyroxine on isolated en- 
zyme systems 188 

Metabolic diseases branch 188 



CONTENTS 



XI 



Page 

Metabolic diseases branch — Continued 

Mineral metabolism 188 

Dietary calcium in osteoporosis 188 

Hormonal and nutritional influences 

on bone metabolism 188 

Human total energy metabolism 189 

Obesity 189 

Energy metabolism in cold ; relation 

to body fat 190 

Blood diseases 190 

Initial stages of blood coagulation . . 190 
Effect of divalent ion chelators on 

blood coagulation 191 

Factor vii 191 

Proteolytic enzyme therapy for intra- 

i. , vascular thrombosis 191 

A new immunologic disease 191 

A new purpuric disease 192 

Antibody reactions in anemia and 

purpura 192 

Pediatric metabolism branch 193 

Cystic fibrosis of the pancreas 193 

Mucus structure and mucopolysaccharide 

metabolism 193 

Electrolyte and genetic studies 193 

Intestinal absorption 194 

Pulmonary involvement 194 

Intestinal malabsorption in children 194 

Glycogen storage disease 194 

National Institute or Mental Health 195 

Basic research 195 

Introduction 195 

The technological challenge 197 

The social challenge 198 

The individual challenge 201 

The natural foundations for human adapta- 
bility 204 

Implications relating to national government 

and diplomacy 213 

General program considerations 216 

The end of the combined basic research pro- 
gram, NIMH-NINDB 217 

Laboratory of neurochemistry 218 

Physical chemistry 218 

Laboratory of neurobiology 219 

Laboratory of neurophysiology 220 

Laboratory of cellular pharmacology 222 

Technical development 225 

Clinical Investigations 227 

Introduction 227 

Clinical care 229 

Adult psychiatry branch 230 

Child research branch 241 

Study of newlyweds 242 

Future directions 248 

Clinical neuropharmacology research center. . 249 

Psychiatry 249 

Chemical pharmacology 251 

Behavioral science 253 



Page 

Laboratory of clinical science 255 

Schizophrenia 255 

Biogenic amines 256 

Fat metabolism and the nervous system. . 257 
Metabolism and inactivation of drugs and 

Hormones 257 

Regional neurochemistry 258 

Thyroxine 259 

Neurophysiology 259 

New techniques 259 

Laboratory of psychology 260 

Child development 261 

Personality 263 

Ofiice of the chief 266 

Animal behavior 268 

Perception and learning 271 

Aging 272 

Laboratory of socio-environmental studies . . . 275 

Social studies in therapeutic settings 276 

Social development and family studies . . . 279 

Community and population studies 281 

Offlce of the chief 283 

Addiction research center 283 

Introduction 283 

Addictive properties of new analgesics 284 

Acute and chronic intoxication with non- 
analgesics, barbiturates or alcohol 286 

Alcohol, barbiturates and related drugs . . 287 

Biochemistry of addiction 287 

Chronic intoxication of barbiturates and • ' 

related drugs 288 

Psychological studies of addiction 289 

Central nervous system depressants 291 

Conditioning factors in addiction and 

habituation 293 

Mental set 295 

National Institute of Neurological Diseases 

and Blindness 297 

Introduction 297 

Medical neurology branch 298 

Neuromuscular disorders 298 

Genetic studies 302 

Brain tumor studies 302 

Epilepsy and cerebral metabolism 303 

Degenerative disorders of CNS 306 

Electroencephalography and clinical neuro- 
physiology branch 306 

Ophthalmology branch 308 

Visual physiology of retinal elements .... 309 

Glaucoma and aqueous formation 313 

Corneal endothelium 316 

Lens structure, physiology, and abnormali- 
ties 317 

Refraction anomalies 319 

Uveal tract inflammations 319 

Neurosurgery branch 320 

Epilepsy and functional anatomy of the 

brain 320 

Involuntary movements 324 



XII 



CONTENTS 



Page 

Neurosurgery branch — Continued 

Stimulation and ablation of CNS by radio- 
frequency energy 324 

Deep hypothermia 325 

Vascular permeability in cerebral edema 325 

Perinatal abnormalities 326 

Laboratory of neurochemistry 327 

Laboratory of neurophysiology 330 

Impulses and nerve excitation 330 

Sensory integrative mechanisms 333 

Electrical changes associated with learn- 
ing responses 333 

Laboratory of biophysics 334 

Voltage clamp techniques 334 

Voltage clamp experiments 335 

Membrane current components and radio- 
active fluxes 335 

Mathematical analysis and computer in- 
vestigations 335 

Acetylcholinesterase Inhibitors and nerve 

activity 336 

Laboratory of neuroanatomical sciences 336 

TJltrastructure of the nervous system . . . 336 

Enzymatic studies on cytologieal fractions 337 

Pathology or artifact 337 

Regeneration and reinnervation 338 

Vestibular and auditory systems 339 

Conclusion 340 

National Institute of Dental Research 343 

Introduction 343 

Laboratory of biochemistry 344 

Collagen and related proteins 344 

Prenatal and dietary factors in experimental 

dental caries 345 

Fluoride studies 345 

Calcification 346 



Page 

Enzyme chemistry 346 

Laboratory of microbiology 347 

Experimental infections 347 

Viral studies 348 

Microbial physiology 348 

Systematic microbiology 349 

Germfree animal research and experimental 

dental caries 349 

Laboratory of histology and pathology 350 

Biophysical studies of calcified tissues 350 

Pathogenesis of dental defects 350 

Enamel surface structure and properties 351 

Histochemistry of connective tissues 351 

Maternal influences on fetal development . . . 352 

Epidemiology and biometry branch 352 

Nutritional factors in oral disease — (preva- 
lence and severity studies in Alaska, Ethio- 
pia, Vietnam, and Ecuador) 352 

Familial factors in oral disease 353 

Clinical investigations branch 353 

Dental caries 353 

Periodontal disease 354 

Calculus formation 354 

Stomatitis 354 

Sjogren's syndrome 355 

Dental pulp 355 

Anesthetic agents 355 

Cephalometric studies 356 

Genetic studies 356 

Division of Biologics Standards 359 

Introduction 359 

Laboratory of control activities 362 

Laboratory of viral immunology 363 

Laboratory of virology and rickettsiology 364 

Laboratory of bacterial products 366 

Laboratory of blood and blood products 367 



NATIONAL CANCER INSTITUTE 



INTRODUCTION 

The staff of the Institute continued to make 
important research contributions of a very high 
order daring 1960. The wide scope of the activi- 
ties, their complexity, and the great numbers of 
important findings make it especially difficult to 
summarize adequately the work, and much addi- 
tional knowledge, often at a rather preliminary 
stage, is not included in the report. Both the 
collaborative groups and the individual scientists 
continue to be highly productive, and the most 
fruitful collaborations are those established by 
the individual scientists themselves finding a com- 
mon meeting ground in research problems of 
joint interest. The attempt to provide a research 
atmosphere most conducive to sound research 
programs must take into account the delicate 
balance between the needs of the Institute as a 
whole and those of the individual. The ideal bal- 
ance, particularly in a large organization with its 
many rules, regulations, clearances and multiple 
channels of communication, is often difficult, if 
not impossible, to attain. Nevertheless, the re- 
search accomplishments made and a sense that 
they are often of high significance for society 
make the considerable effort directed toward this 
end worthwhile. 

At mid year, a number of changes occurred in 
several key positions in the Institute. Dr. Ken- 
neth M. Endicott assumed the position of Direc- 
tor, NCI, following two years as Associate Direc- 
tor, NIH. Dr. C. Gordon Zubrod, who has been 
the Clinical Director of the NCI since 1954, as- 
sumed responsibility for the clinical programs, 
including the Surgery, Endocrinology, General 
Medicine and Radiation Branches. Dr. Carl G. 
Baker, who had been Assistant Director with 
Dr. Heller for 2y 2 years, assumed responsibility 
for the nonclinical aspects of the intramural pro- 
gram, including the Laboratories of Biochemis- 
try, Biology, Physiology, Chemical Pharmacol- 
ogy and Pathology, and the Pathologic Anatomy 
Branch. 

These organizational changes and the impend- 



ing retirement of Dr. Howard B. Andervont as 
Chief of the Laboratory of Biology brought about 
extensive discussions of the direction the Insti- 
tute should take, of the areas of emphasis and 
de-emphasis, and of intraorganizational relation- 
ships. One change is the expectation that scien- 
tists on the intramural staff will participate more 
actively in some of those programs, formerly 
classified as "extramural" or in the "gray area," 
for which the Institute staff has clear responsi- 
bility as regards quality and execution. In the 
future, it appears this will be of special impor- 
tance in the areas of international medical re- 
search and, under Field Studies, cancer diagnostic 
test development and environmental cancer. The 
major program areas which in the near future 
will receive most attention throughout the Insti- 
tute are: viruses and cancer; molecular and de- 
velopmental biology; environmental cancer; and 
cancer chemotherapy. The highly competitive 
market for senior scientific personnel in these 
areas makes it difficult to proceed as rapidly as 
one might wish, and availability of facilities also 
sets limits on the rate of development of program 
segments. 

The relationships with the Board of Scientific 
Counselors continue to be excellent, and the pres- 
ent Board, made up of Drs. Philip P. Cohen, 
Chairman, Wendell M. Stanley, E. K. Marshall, 
Jr., Hugh Roland Butt, Jacob Furth and J. En- 
glebert Dunphy, exhibits the same high interest, 
helpful advice and general support as has be- 
come customary. At the April meeting of the 
Board, a program of research studies by the 
Clinical Associates was presented by these young 
men, and at the fall meeting, a program was held 
on broad aspects of cancer research, its state of 
development, its future coast, and the strategy 
involved. The Laboratory and Branch Chiefs 
discussed their programs as they relate to these 
broad subjects. 

The Board continues to express confidence in 
the research staff and its programs. The frank 
discussions characteristic of past meetings con- 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tinued, and selected problems facing the Institute 
were discussed with a free exchange of ideas and 
suggestions. Not only do the members of the 
Board provide the Laboratory and Branch Chiefs, 
and, indeed, individual staff members, the oppor- 
tunity to discuss scientific and organizational 
problems with them, but as disinterested parties, 
very real assistance is given to Dr. Endicott and 
his immediate staff. 

The NCI was visited by a delegation of out- 
standing cancer investigators from the Soviet 
Union, September 26-28, 1960, in accordance with 
implementation of the Lacy-Zarubin agreements 
and subsequent detailed plans developed between 
the Public Health Service and the Ministry of 
Health, USSR. The group consisted of: 
Professor N. N. Blokhin, Director of the Insti- 
tute of Experimental Pathology and Therapy 
of Cancer, Moscow, President of the Academy 
of Medical Sciences, USSR, and Head of the 
Delegation. 
Dr. L. A. Zilber, Director, Department of Im- 
munology and Malignant Tumors and Scien- 
tific Director, Institute of Epidemiology and 
Microbiology in the name of Gamalei, Mos- 
cow. 
Dr. L. F. Larionov, Head, Laboratory of 
Experimental Chemotherapy, Institute of 
Experimental Pathology and Therapy of 
Cancer, Moscow, and an editor of "Patho- 
logicheskaya fiziologiya i experimentalnaya 
terapiya." 
Dr. M. M. Mayevskiy, Head, Laboratory of 
Experimental Biotherapy, Institute of Ex- 
perimental Pathology and Therapy of Can- 
cer, Moscow, and an editor of "Zhurnal 
mikrobiologii epidemiologii i immunobiolo- 
gii." 
V. V. Gorodilova, Candidate in the medical 
sciences, Head, Laboratory of Virology and 
Acting Director, Central Institute of Oncol- 
ogy in the name of P. A. Gertzen, Moscow. 
Cordial relationships were maintained through- 
out the visit, and since the main interests of the 
visitors were virology and cancer, immunology 
and cancer chemotherapy, most of the presenta- 
tions by NCI staff members were in these scien- 
tific areas. Little new information on cancer re- 
search in the Soviet Union was obtained from 
the visiting scientists. However, two talks were 
presented: Dr. Larionov spoke on "Complex 



Alkylating Metabolites as a New Type of Anti- 
tumor Drug" and Dr. Zilber spoke on "Suscep- 
tibility of Rats and Rabbits to Rous Sarcoma 
Virus." It is planned that a group of American 
cancer investigators will make a return visit to 
the Soviet Union in May of 1961. This group 
will probably consist of Drs. Endicott, Ander- 
vont, and Hertz from NCI, and from the Sloan- 
Kettering Institute, Drs. Horsfall, Stock, Dall- 
dorf and Randall. As part of the program for 
the exchange of individual scientists who will 
spend longer periods of time working in labora- 
tories, Dr. J. Edgcomb may spend 6 months or 
longer in the Soviet Union, and two Soviet scien- 
tists are scheduled to spend a year at the Sloan- 
Kettering Institute. 

Several members of the staff have received out- 
side recognition for their accomplishments. Dr. 
Murray Shear is currently President of the Amer- 
ican Association for Cancer Research and Chair- 
man of the Finance Committee and of the Chemo- 
therapy Committee of the International Union 
Against Cancer. Dr. H. L. Stewart is Chairman 
of the Geographic Pathology Committee of the 
Union as well as a member of its Executive Com- 
mittee. Dr. Thelma Dunn is Vice President and 
President Elect of the American Association for 
Cancer Research, assuming the office of President 
next year. Dr. Roy Hertz is Vice President of 
the Endocrine Society. Dr. Eugene Van Scott 
is a member of the Board of Directors of the 
American Academy of Dermatology and Syphil- 
ology. Drs. Herbert A. Sober and Carl G. Baker 
are members of the Executive Committee of the 
Division of Biological Chemistry, American 
Chemical Society, and Dr. Baker represents the 
Division as one of two Divisional Councilors on 
the American Chemical Society Council. Dr. 
Harold F. Blum received a Sigma Xi award for 
his article "Quantitative Aspects of Cancer In- 
duction and Growth: as Illustrated in Carcino- 
genesis by Ultraviolet Light," American Scien- 
tist 47, 250-260 (1959). Drs. John Weisburger 
and Vincent E. Price became members of the 
Editorial Board of "Cancer Research." Dr. A. J. 
Dalton was elected a Director of the Electron 
Microscope Society of America and was ap- 
pointed to the Council of the American Associa- 
tion for the Advancement of Science. Many 
members of the staff continued to give invited 



NATIONAL CANCER INSTITUTE 



lectures and serve in consultative capacities 
throughout the country. 

As an honor to Dr. Wilton Earle and his asso- 
ciates, the Tissue Culture Section was asked by 
the Cell Collection Coordinating Committee to 
supply clone NCTC 929 of Strain Z mouse fibro- 
blasts as the first selection to go to the repository 
for characterized tissue culture cell lines of the 
American Type Culture Collection. 



CLINICAL INVESTIGATIONS 1 

The clinical branches of the intramural research 
effort of the National Cancer Institute have en- 
joyed a productive year. This is attested to not 
only by the high quality of research publications 
on major problems of clinical cancer, but also by 
continual calls upon the staff for advice, invited 
lectures and service upon national committees 
related to cancer research. In addition to the 
honors listed above, a few others should be men- 
tioned. Dr. Koy Hertz was called upon to advise 
Secretary Flemming on problems of carcinogene- 
sis. He was also elected to the American College 
of Physicians. Dr. Nathaniel Berlin was elected 
to the American Society for Clinical Investiga- 
tion. Dr. John Fahey spent a year as a visiting 
scientist at the National Institute for Medical 
Research, Mill Hill, England. Dr. Mortimer 
Lipsett is now a visiting scientist at the Karo- 
linska Institute. Dr. Kurt Kohn is working for 
a period of several years at Harvard with Dr. 
Paul Doty. 

There have been a number of changes among 
the senior staff. Dr. Donald Watkin, General 
Medicine Branch, after nine years at NIH, left 
for a year's work with the Institute of Nutrition 
of Central America and Panama, surveying nu- 
tritional problems of the Caribbean. He will 
soon assume his new post of Associate Professor 
of Nutrition at Massachusetts Institute of Tech- 
nology. Dr. Montague Lane, General Medicine 
Branch, became Assistant Professor of Pharma- 
cology at Baylor University. Dr. Robert Mad- 
den, Surgery Branch, left to become Assistant 
Professor of Surgery at New York Medical 
College. 

Dr. Thomas Butler, Professor of Pharmacol- 



i Prepared by C. G. Zubrod, M.D., Clinical Director. 



ogy, University of North Carolina, spent eight 
months as visiting scientist with Dr, David Ball 
working on problems of intracellular pH. Dr. 
Tatuo Saito, Associate Professor of Internal 
Medicine at Tohoku University Medical School, 
Sendai, Japan, completed a year as guest worker 
at the National Cancer Institute. He worked 
with Dr. David Rail on the transfer of glucose 
from blood to the central nervous system. Dr. 
Myron Karon joined the Chemotherapy Service 
as the first full time pediatrician at NCI. Dr. 
Paul Carbone transferred from the Hospital Di- 
vision, to the Chemotherapy Service. Dr. Sher- 
man Weissman, a former clinical associate, re- 
turned as a senior staff member of the Metabolism 
Service. Dr. Jacqueline Whang has accepted a 
fellowship on the Chemotherapy Service for cyto- 
genetic studies of leukemic patients. The Radia- 
tion Branch has Dr. Roger Berry and Dr. Law- 
rence Draper as new staff members. Dr. Berry is 
working on the biological effect of neutrons, and 
on chemical enhancement of irradiation. Dr. 
Draper's interests concern immunological phe- 
nomena as altered by irradiation. Two new 
senior members have joined the Surgery Branch: 
Dr. John T. West, who transferred from the 
Hospital Division, and Dr. Everard Cox, who 
came from the University of Maryland. In the 
Endocrinology Branch, Dr. Griff T. Ross has ac- 
cepted major responsibilities after a number of 
years at the Mayo Clinic, and Dr. William Odell 
has joined the laboratory group working on pitui- 
tary hormones. 

These yearly comings and goings of senior staff 
raise the questions of what it is that attracts 
clinical investigators to NIH, why some elect to 
stay, why some leave. One of the obstacles to 
full staffing is posed by the nature of the "Cancer 
Hospital." Some types of research can be justi- 
fied only in those patients whose disease is not 
amenable to cure. Thus many of our patients 
have relatively far advanced cancer, often with 
the stresses of pain and physiological impair- 
ment, occasionally disfigured from previous sur- 
gery, with depleted nutritional and emotional 
reserves, and death often not far away. One or 
two of these patients can almost paralyze the 
usual medical service. Our staff must be prepared 
to care for over a hundred such patients. To do 
so is costly — in resources, in personnel, in de- 
mands upon the moral courage of our staff. For 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



such care we must have physicians who are com- 
passionate, skillful, wise and yet good investi- 
gators interested in patients with cancer. Combi- 
nations such as these are uncommon, perhaps rare. 
There are 8,0004- graduates from medical schools 
in this country each year. NIH receives inquiries 
from about 1,500; 400 are considered seriously; 
100 are interviewed by NCI; 18 are appointed 
as Clinical Associates. From each eighteen in 
some years, we find one who is qualified to stay 
on. For these physicians and for all our staff, 
efforts of the Institute must be intensified to make 
their careers here rewarding, effective and satis- 
fying. 

The content of clinical investigation of cancer 
requires one comment. No useful purpose would 
be served by attempting to study all phases of 
human cancer, just as a century ago it would have 
been fruitless to arrive at an understanding of 
virus diseases by studying intensively the mech- 
anisms and control of fever. The leads in cancer 
research must come from a vigorous nonclinical 
program. It is the task of the clinician to iden- 
tify in the clinic those situations which will per- 
mit him, in a highly controlled fashion to validate 
the leads of the laboratory scientist. The clinical 
program is based on this assumption and the re- 
view of the past year's efforts provides many 
examples. The review will be divided into three 
sections : Origin of the Cancerous State, Develop- 
ment of Clinical Cancer and Management of 
Cancer. 

I should like to call attention to several devel- 
opments which can be regarded as of major 
importance: 

The description by Dr. Roy Hertz and his 
colleagues of 5-year survivals of patients 
with metastatic trophoblastic disease follow- 
ing methotrexate treatment. 

The development by Dr. Eugene Van Scott 
of ways to study cellular differentiation in 
man. 

The definition by Drs. Robert Smith and 
John T. West of the limitations of sampling 
of tumor cells in wounds and peripheral 
blood. 

The development by Dr. Emil Freireich 
and Dr. Allan Kliman (DBS) of a practical 
way to treat thrombopenic hemorrhage by 
platelet replacement without the development 
of immunity. 



The emergence of chemotherapy studies 
under Drs. Frei, Rail and Goldin into a new 
phase which allows study of the specificity of 
animal tumor systems for prediction of drug 
activity for particular tumors of man. 

The maturation of the NCI registry of 
patients to the level that all major studies 
report 100 percent followup. 

The recognition by Drs. Ketcham, Hoye, 
and Berlin of the magnitude of blood loss 
during and following surgery, and the fail- 
ure of the hematocrit to provide a guide for 
full replacement. 

The demonstration by Drs. J. R. Andrews 
and Brace that radiation therapy of some 
cancers can be safely and adequately given 
in a single dose. 

Origins of the Cancerous State 

There is now a large body of opinion which 
holds that a normal cell becomes a cancer cell 
because of some damage to the DNA replicating 
mechanism. This damage can apparently be initi- 
ated by a variety of "causal factors" such as 
irradiation, chemicals, genetic defect, viruses or 
metabolic abnormality. It has been difficult for 
the NCI intramural clinician to identify clinical 
situations worth study. Most of the leads come 
from epidemiological considerations, and here our 
clinicians have had few opportunities. Perhaps 
the newly formed Field Studies Group under Dr. 
Shimkin will create the openings for the clinician 
to study carcinogenesis. (One such opportunity 
developed recently when the uranium miners' 
study revealed eight men with suspicious cytol- 
ogy, but lack of adequate surgical staff made ad- 
mission unwise.) 

Another reason for inability to initiate program 
has been the status of the art of uncovering onco- 
genic viruses. Drs. Manaker, McLaughlin, and 
Couch studied 27 primary tumors from NCI pa- 
tients, by tissue culture techniques, designed to 
unmask such viruses, without positive result. 
Vigorous pursuit of human oncogenic viruses 
awaits new discoveries on how to uncover them. 

Several illustrations may serve to emphasize 
how indirect must be the approach of the clini- 
cian to carcinogenesis. Drs. Van Scott, Reinert- 
son, Eisen and Rothberg studied patients with 
psoriasis as a model of skin hyperplasia, and tried 



NATIONAL CANCER INSTITUTE 



to relate the hyperplasia to biochemical abnor- 
mality. Recognizing psoriasis as a systemic ab- 
normality with arthritis and hyperuricemia they 
have shown in work with Dr. Seegmuller, NIAMD, 
that in this disease C-14 glycine is over-incorpo- 
rated into uric acid; that the uric acid pool is 
increased in size, not to the degree seen in some 
cases of gout, but generally greater than that 
noted in chronic myelocytic leukemia. Drs. Roth- 
berg and Van Scott have also shown that C-14 
labelled glycine has a much more rapid turnover 
time (3-5 days) after incorporation into protein 
of psoriatic scales, than after incorporation into 
normal skin (28 days). The reasons for these 
phenomena are unknown, but the model exists for 
further study of the relation of hyperplasia to 
disturbances of nucleoprotein and protein metab- 
olism. 

The observations of Dr. John L. Fahey on 
plasma-cell tumors in man provide another pos- 
sible approach to clinical carcinogenesis. Con- 
tinued work with Dr. Michael Potter on eight 
plasma-cell tumors of mice, showed that the pro- 
teins formed by these cells have distinctive 
electrophoretic, ultra-centrifugal and immuno- 
logic properties. The individuality of the my- 
eloma proteins suggests that each neoplasm arose 
from a single plasma cell clone. This observation 
is in keeping with those of Morris and Van Potter 
and of Tomkins on the enzymatic activities of 
rat hepatomas. Dr. Fahey's work on plasma cell 
tumors in man shows similar specificity and indi- 
viduality of proteins and thus the clinician is 
provided with a marker by means of which the 
behavior of a malignant cell clone can be inferred. 
In this manner some of the many new observa- 
tions on the individuality of chemical activities 
of induced tumors can perhaps be tested in the 
clinic. 

Another way in which carcinogenic influences 
in man can be followed is by means of cytogenetic 
changes. Qualitative and quantitative abnormali- 
ties of chromosomes have been described as genetic 
faults, as a result of radiation and in association 
with tumors. Drs. Hertz and Ross have used 
chromosomal counting as a tool for identification 
of various genetic growth abnormalities in pa- 
tients which can then be characterized endocrino- 
logically. Dr. Jacqueline Whang has recently 
joined the staff and is studying cytogenetic ab- 
normalities in the leukemias and in mongolism. 



In collaboration with Dr. Tijo of NIAMD, she 
has simplified the methods for chromosomal mor- 
phologic study and enumeration, permitting more 
extensive survey of changes in disease. 

Development of Clinical Cancer 

Once established, cancer cells invade and de- 
stroy locally, spread into distant tissues and in 
the process of growth produce varying effects 
upon the host. The mechanisms of these processes 
may be studied in the clinic from the following 
aspects : 

1. autonomy of cancer cells; 

2. the spread of cancer cells from the point 

of origin to remote sites, where they 
become established and grow; 

3. the resultant physiologic and biochemical 

abnormalities. 

Autonomy 

By autonomy, one means the property of can- 
cer cells by which they no longer respond to those 
controls which the host normally exerts over 
cellular proliferation, differentiation, and inva- 
siveness. In the normal situation, for example, 
the cells of the vermilion border of the lip, al- 
though multipotential, remain throughout life as 
a sharply defined line. Somehow the body relays 
information to these cells to remain as mucous 
membrane. Drs. Van Scott and Reinertson have 
shown that buccal mucous membrane, when trans- 
planted autologously to a skin area, retained its 
characteristics only when its connective tissue 
stroma was still attached. When pure mucosa 
was transplanted subepidermally, it became kerat- 
inized epidermis; when placed in the lower co- 
rium, it developed ductlike structures. The same 
pattern was observed after the transplantation of 
other skin components such as hair roots. These 
observations have been extended to basal cell car- 
cinoma. Pure basal cell cancer implanted into 
the lower corium degenerated, keratinized or 
formed ductlike structures. When transplanted 
subcutaneously, the tumor always degenerated. 
The authors conclude that although the range 
of responses possible for epithelial cells appears 
to be a property of the cell line, the specific re- 
sponse is determined by the environment in its 
local connective tissue stroma. They have further 
attempted to define the environmental influences 



6 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



leading to keratinization by tissue culture study 
of epidermal cells. Keratinization is enhanced by 
the elevation of the pH of the culture medium 
above normal or the presence of high C0 2 tension. 
Thus, though the environmental control may be 
exerted through complex chemical influences, it is 
also possible that in vivo the control may be 
physical in nature. Drs. Van Scott and Reinert- 
son have developed a model for the study in man 
of cellular differentiation of both normal cells 
and cancer. 

Autonomy can also be subjected to clinical scru- 
tiny in the classic situation of the study of hor- 
monal control of sensitive tissues. Dr. Roy Hertz 
and his colleagues have continued their observa- 
tions on the mechanisms by which hormones limit 
or enhance the growth of normal cells and cancer. 
Drs. Hertz, Griff T. Ross and Mortimer Lipsett 
in a study of five men, found breast cancer to be 
remarkably sensitive to orchiectomy. Four of 
these, after recurrence, responded to estrogen ther- 
apy. There were no abnormalities of thyroid, 
pituitary or adrenal function. Thus they have 
identified another highly sensitive system for 
studies of hormonal control. In studies of breast 
cancer in women, they have found neither exacer- 
bation or regression of disease after the adminis- 
tration of human or sheep follicle stimulating 
hormone, human growth hormone, or ovine pro- 
lactin. The latter two materials caused no exac- 
erbation of prostatic cancer. Growth hormone 
was studied extensively for its metabolic effects. 
It did not cause nitrogen retention in four pri- 
mordial dwarfs. Reports of others on the activity 
of chymotrypsinogen treated beef growth hor- 
mone could not be confirmed. In addition, al- 
though testosterone caused nitrogen retention in 
active acromegaly, growth hormone did not. Dr. 
J. M. Van Buren, NINDB, in a collaborative 
report with the late Dr. Delbert Bergenstal, eval- 
uated 12 patients with breast cancer treated by 
hypdphysectomy. Seven of the 12 had remissions 
lasting 3-9 months. Serial sections of the hy- 
pophysis at autopsy indicated the degrees of 
completeness of pituitary removal. Twenty-nine 
to 0.01 percent of the gland remained. Neither 
the degree of depression of endocrinologic func- 
tion nor the extent of regression of breast cancer 
was in any way correlated with the residual 
amount of pituitary. If less than 10 percent of 
the gland remained, the patients were perma- 



nently cortisone dependent. On the other hand 
pituitary stalk section in another 12 patients with 
breast cancer showed little effect upon tumor, and 
several patients showed persistence of thyroid 
function. 

In studies of the hormonal control of prostatic 
tissue, Dr. William W. Tullner has shown in 
castrate male rats that ACTH or ACTH plus 
prolactin produced growth of the ventral prostate 
only in the presence of the adrenals and hypophy- 
sis. He concludes that pituitary growth is, in 
part, dependent not only on adrenal androgens 
but also on unidentified pituitary factors. 

Spread of cancer 

Studies in man by several investigators includ- 
ing Dr. Robert Smith and his colleagues at NCI 
in collaboration with Dr. R. S. Malmgren and 
the staff of the Cytodiagnostic Service, have re- 
vealed that microscopic fragments and even sin- 
gle cells from a cancer can be found in wound 
washings after surgery or in the circulating blood. 
The recent report by Dr. George Moore that 
ThioTEPA given at the time of radical mastec- 
tomy has substantially reduced recurrences com- 
pared to controls, emphasize that an understand- 
ing of tumor spread is the most important 
problem facing surgical research. Continued 
study of cellular detachment, spread and growth 
in patient and animal during the past year has 
helped further to define the problem. 

Wound Washings. Drs. Smith and Marvin 
Arons have followed 100 percent of 111 patients 
who had excision of primary cancer between 
September 1953 and December 1956. The fol- 
lowup period since operation was 44 to 83 months. 
Twenty-six percent of the patients had positive 
wound washings; 60 percent were negative and 
14 percent suspicious. There were no correlations 
between the results and local recurrence, lymph 
node or distant metastases, or survival. The lack 
of prognostic significance of positive wound wash- 
ings might well be due to sampling problems. 
Dr. Seymour C. Nash has attempted to obtain 
more complete wound samples by inserting a suc- 
tion catheter into the wound for several days. 
At intervals, samples of the wound washings, 
trapped and iced in heparin and streptodornase- 
streptokinase, were removed for counting. Tumor 
cells were demonstrable in post-operative drain- 



NATIONAL CANCER INSTITUTE 



age fluids as long as 43 hours after operation, and 
sometimes when the washings at the time of oper- 
ation were free of cells. 

Cancer Cells in Circulating Blood. Although 
other investigators have found no correlation be- 
tween cells in the circulating blood and progno- 
sis, it is doubtful whether adequate experimental 
design of a study has yet been attained in man. 
Dr. John T. West and Dr. Malmgren have con- 
tinued to validate methodology, and still face 
problems of identification of cells. An equally 
serious problem is that of sampling, as shown in 
their study of 377 patients, 26.3 percent of which 
were positive for circulating tumor cells. There 
was a linear relation between the number of ex- 
aminations and the percentage positive. Of 183 
patients with one examination, 10 percent were 
positive. Percentage positives increased with re- 
peated examinations until, with seven or more 
examinations, 75 percent of 45 patients were posi- 
tive. Prognostic significance cannot be judged 
until sampling comparability is achieved. It can 
be inferred that the present technique is less than 
10 percent efficient. The limiting factor is prob- 
ably the small amount of blood which can be 
processed with present techniques. If a method 
can be found permitting greater efficiency of re- 
covery, then it will be possible to examine the 
influence of manipulation, exercise, and other fac- 
tors in releasing cells into the circulation. One 
modification in the millipore technique has been 
introduced by Dr. Marvin Komsdahl who substi- 
tuted dextran sedimentation for lysis of erythro- 
cytes. This permitted the adaptation of quanti- 
tative recovery from blood of the cells of various 
mouse tumors. 

Spread of Tumor in Animal Models. The ani- 
mal models of tumor spread developed by mem- 
bers of the Surgery Branch have been ably uti- 
lized by Dr. Alfred Ketcham and his colleagues. 
Dr. Romsdahl injected Sarcoma T-241 tumor 
suspensions into the hind leg of C-57 mice, ampu- 
tated the left leg on the next day in one group 
of animals and on succeeding days in other 
groups. He found that although circulating 
tumor cells could be found 24 hours after inocu- 
lation, amputation before the seventh postopera- 
tive day prevented pulmonary metastases. He 
concluded that the demonstration of tumor cells 



in blood is not necessarily equivalent to hema- 
togenous metastases. 

Last year it was reported by Dr. David Kinsey 
that certain tumors will metastasize only to pul- 
monary tissue, even if the lung is transplanted 
away from the pulmonary circulation. Dr. Mar- 
vin Arons found that metastasis did not occur to 
a similarly implanted etheron sponge in DBA/P 
mice with L1210 leukemia, S-91 melanoma or a 
mast cell tumor. Even though connective tissue 
would rapidly enter the sponge, tumor would 
grow around but not infiltrate it. 

A number of host factors which influence me- 
tastases have been recognized. Dr. Ketcham 
transplanted several mouse tumors into mice of 
different ages and found that the growth of the 
primary tumor was delayed in the older mice. 
Similar delay was noted if the fluid intake of the 
mice was limited to 20 percent ethanol, but there 
was no effect from denervation of the limb into 
which the tumor was transplanted. Dr. Alan 
Eetik showed an increase in metastases of S-91 
melanoma to the previously traumatized liver. 

Previous reports have emphasized the failure 
of drugs to modify the local growth or metastases 
of transplanted tumors. In the past year Dr. 
Ketcham has shown accentuation of growth of a 
variety of transplanted mouse tumors when drugs 
such as clorpactin, thioTEPA or alcohol were 
placed in the wound before the inoculum. When 
the order was reversed, there was a delay in 
growth related entirely to the mechanical effects 
of washing, except with 3,6-diamino acridine. Dr. 
Nash reported that with a mast cell tumor, this 
agent locally applied 1 to IV2 hours after tumor 
inoculation reduced takes to 4 percent whereas 
saline washed controls had 100 percent take. 
Similar results were obtained with S-91 mela- 
noma. This is an interesting lead because of the 
variety of acridines available and their pharma- 
cological property of adsorption to nucleopro- 
teins. 

Effects of Cancer Upon the Patient 

Clinical cancer has taken the place of syphilis 
as "The Great Imitator," for it can appear in 
every conceivable guise. This has always been 
true but has only recently become fully apparent 
as the patient with cancer has been recognized 
as a sick human being, rather than a technical 



8 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



problem in early diagnosis, surgery, radiation or 
referral to a nursing home. The bewildering 
kaleidoscope of clinical cancer is in large part due 
to the capacity of cancer to infiltrate any organ, 
with resultant change in function, either as a 
single organ deficit or in unending combinations 
with damage to other organs, susceptibility to 
infection and emotional response. The tempta- 
tion to study intensively these clinical fireworks 
is difficult for the good physician to resist. And 
this is fortunate for it leads to precisely the kind 
of intimate, compassionate and sophisticated care 
which is the right of every patient and the duty 
of each physician. It should be appreciated that 
this "research," vital as it is to the care of the 
patient and to the continued development of good 
doctors, contributes little to the understanding of 
cancer per se. This is perhaps not wholly true 
since some organ destruction of general character, 
such as of bone marrow in the leukemias or of 
lymphoid structures in the lymphomas, may re- 
late to the nature of the disease. 

In addition to the direct effects of infiltration, 
cancer produces a number of indirect responses 
remote from the site of tumor cells. Many of 
these indirect effects are brought about by chemi- 
cal substances such as hormones manufactured 
by the cancer, and even though these do not bear 
any more relation to the nature of cancer than 
anatomic and physiologic alterations, they do 
provide ready-made labels by which tumor be- 
havior can be followed quantitatively. In addi- 
tion, there are indirect effects of unknown causa- 
tion, such as nutritional depletion of the host 
which, if understood, might give clues to the 
mechanisms of autonomy. 

These are the assumptions upon which we have 
based the clinical investigation of the behavior 
of cancer. 

Direct Effects of Cancer Growth 

Blood Forming Tissues — Platelets. The inten- 
sive study of mechanism of hemorrhage in the 
leukemias has continued. It has always been 
puzzling why some patients with marked throm- 
bocytopenia should show no bleeding tendency 
and the question has remained as to the need to 
postulate a pathogenetic factor other than platelet 
deficiency. Drs. E. J. Freireich, L. R. Schroeder 
and L. Gavdos have been able to demonstrate in 



92 leukemic patients with hemorrhage a direct 
relationship between degree of thrombocytopenia 
and risk of hemorrhage. Working in collabora- 
tion with Dr. A. Kliman (DBS), a technique has 
been developed for the separation and concentra- 
tion of large numbers of platelets. The technique 
utilizes repetitive plasmapheresis of single donors. 
Twenty leukemic children with severe thrombo- 
penia have been treated with these preparations; 
nineteen showed response with clinically signifi- 
cant increase in circulating platelets and control 
of hemorrhage. Contrary to expectation, no in- 
stance of immunization was seen even though 
patients received repeated platelet transfusions 
over a period of several months. The investi- 
gators feel that if larger amounts of platelets can 
be given and thus the treatment made available 
to adults, the way may be open to the more com- 
plete control of thrombocytopenic bleeding. 

Dr. Schroeder has continued his search for a 
"platelet hormone" and has defined some of the 
conditions for platelet control in rabbits. 

Leucocytes and Immunity. The effects of dif- 
fuse cancer upon leucocytes are quite complex 
because of the variety of cell types and functions 
arising in marrow and lymphoid tissues. The 
interference in leucocyte production is manifested 
largely by an increased susceptibility to infection. 
Studies by Drs. Frei, Paul Carbone and Fahey 
with the collaboration of Dr. J. P. Utz (NIAID) 
have helped to unravel some of the mechanisms 
of diminished host resistance. Thus, infections 
in patients with acute leukemias were shown to 
be due to rapid depletion of polymorphonuclear 
leucocytes without alterations in antibody pro- 
duction. Patients with chronic lymphocytic leu- 
kemia were shown to have frequent and severe 
infections which correlated well with inability to 
respond to antigenic challenge but only partially 
with the degree of hypogammaglobulinemia. In 
a controlled comparison, it was shown that pred- 
nisone treatment of chronic lymphocytic leukemia 
increased the frequency and severity of infections, 
gave no systematic anti-leukemic response and is 
contraindicated except for management of hemo- 
lytic anemia. Susceptibility to infection in other 
lymphomas seems unrelated to either above factor 
and is in some way related to inability to combat 
virus infections, as in seven adults noted with 
autopsy evidence of cytomegalic inclusion disease. 



NATIONAL CANCER INSTITUTE 



Dr. Fahey's approaches to the isolation and 
characterization of antibodies may help to inves- 
tigate some of the above problems. Working 
with antithyroglobulin and antiliver nucleopro- 
tein antibodies, he has shown that some antibodies 
were solely among the 6.6 S gammaglobulins, 
some were 18 S and some were present in both. 

Gastrointestinal Tract. In addition to varied 
globulin disturbances, hypoalbuminemia is com- 
mon in patients with cancer. Dr. T. Waldmann, 
in collaboration with Dr. E. Gordon (NHI), has 
developed techniques for studying the pathogene- 
sis of serum protein abnormalities. These have 
been applied to the elucidation of hypoalbu- 
minemic states relates to gastrointestinal disease 
as previously reported. New findings relate to 
hypoalbumineria in lymphoma. Study of 15 pa- 
tients with lymphoma and hypoalbuminemia 
showed reduction of synthetic rate of albumin in 
all. Albumin survival was prolonged in two 
patients with effusions, while survival was short- 
ened in the remaining 13. Albumin loss into the 
gastrointestinal tract was not observed. 

Bone Metastases. The behavior of metastases 
in bone can be a valuable guide to the advance or 
regression of cancer. Unfortunately, changes in 
X-ray lag months behind biological behavior. 
Dr. L. Avioli has used Ca-47 in association with 
external scanning and metabolic balance studies. 
He has confirmed the marked elevation of bone 
formation and resorption in patients with lytic or 
blastic lesions. The external scanning with Ca- 
37 seemed to reflect regression or progression 
months in advance of the corresponding X-ray 
change. 

Indirect Effects of Cancer Growth 

The production in the host of chemically me- 
diated effects is classically illustrated by endo- 
crine active tumors, myeloma and carcinoid. In 
addition, there are a number of instances of 
apparent remote effects where no chemical prod- 
uct has been found and the obverse, in one in- 
stance, of a chemical product with no physio- 
logical disturbance. 

Hormonal Products of Cancer. The endocrine 
program of Dr. Roy Hertz and colleagues en- 
compasses many aspects of this problem. The 



resourcefulness of these investigators is shown in 
the development of quantitative assay procedures, 
application to the study of hormonal physiology 
in animals, and rapid extension to disease prob- 
lems. Examples of this may be seen in their 
studies of adrenal cortical hormones. Dr. M. 
Lipsett, with the collaboration of Dr. H. Wilson 
(NIAMD), has developed methods for the isola- 
tion and quantitation of a wide variety of steroid 
precursors and end-products. Application of 
these procedures to patients with functioning 
adrenal cortical cancer has shown that in all pa- 
tients there was an elevated excretion of delta-5- 
pregnenetriol, which percentagewise was greater 
than the increase in dehydroepiandrosterone. As 
a result of further analysis of the possible points 
of enzymatic block, they found that 11-beta- 
hydroxylation was generally impaired, and in 
two instances the block was complete. Preg- 
nenetriol was excreted in elevated amounts in 
many of the patients, both with adrenal cancer 
and with hyperplasia. They also concluded that 
21-hydroxylation was variably impeded and 50 
percent of patients with adrenal cancer lacked 
3-beta-hydroxydehydrogenase. In studies of the 
increase in dehydroepiandrosterone, they found 
that the desmolase could not dispose of the 
amounts of the precursor, 17-hydroxypregneno- 
lone, presented to it; nor could they show that 
the increased dehydroepiandrosterone arose from 
exogenous cholesterol. Excess etiocholanalone 
excreted by adrenal cancer patients was shown 
to have unidentified precursors, other than an- 
drostenedione and dehydroepiandrosterone. 

Drs. Lipsett and L. Garren examined a num- 
ber of hypermetabolic euthyroidal patients with 
leukemia or lymphoma. Cortisol was metabolized 
at a normal or slowed rate, but Cortisol secretion 
was increased. They concluded that the effect of 
hyperthyroidism on Cortisol metabolism is due to 
a specific action of thyroxine and is not related 
to hypermetabolism per se. These techniques are 
also proving helpful in understanding the com- 
plexities of endocrine relationships in patients 
with pituitary tumors, hypophysectomy, genetic 
growth abnormalities and Cushing's syndrome 
associated with tumors of nonendocrine origin. 

Dr. Hertz has made a number of observations 
on another chemical product of tumors — chori- 
onic gonadotropin in patients with trophoblastic 
tumors. In the women with tumor, he found a 



10 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



normal pregnandiol excretion indicating that the 
luteal phase of ovarian function was unaltered. 
Four patients with high gonadotropin excretion 
had normal menstrual cycles, reflecting lack of 
interference with normal pituitary gonadal rela- 
tions. Not only does chorionic gonadotropin of 
pregnancy prolong the luteal phase of the normal 
cycle, it will stimulate the interstitial cells of the 
testes of hypogonadal males. In two such boys, 
the interstitial cells were stimulated by pure 
gonadotropin from patients with trophoblastic 
tumors. He concluded that the refractoriness of 
the ovaries in the tumor-bearing patient involves 
some special mechanism for interference with 
peripheral gonadal action. This refractory state 
appears late in the course of trophoblastic dis- 
ease; at an earlier phase the ovaries show con- 
siderable enlargement. 

Another difference between placenta and tro- 
phoblastic tumor has been found by Dr. D. Kel- 
logg. Tumor tissue contained only a minute 
fraction of the steroid dependent transhydro- 
genase and dehydrogenase found in normal pla- 
centa.' He also found by histoehemical techniques 
that each enzyme had a different locus in placen- 
tal cells, supporting the view that they are differ- 
ent enzymes. 

Dr. S. Genuth has devised a method for the 
detection of minute amounts of gonadotropin in 
the rat hyperemia test. This assay is now ap- 
plicable to the study of substrate influence on 
synthesis of gonadotropin by cell free systems. 

An annoying fuzziness in gonadotropin assays 
resulted in findings which illustrate the ability 
of Dr. Hertz and his colleagues to capitalize on 
chance ? as well as the importance of resolving 
discrepancies. Unexplained uterine stimulation 
in immature mice led to the discovery by Drs. 
Hertz and Tullner that an insecticide, used for 
ectoparasite control, was responsible. The insec- 
ticide proved to be a mixture of Methoxychlor 
isomers, analogues of the adrenocortical inhibitor, 
DDD. Further work showed that the mixture 
was uterotrophic for the mouse, even in the ab- 
sence of adrenals or anterior pituitary, and that 
in parabiotic rats it inhibited the gonad stimu- 
lating effect of pituitary gonadotropin. It also 
stimulates testicular growth in male rats, but has 
little effect on the adrenal cortex of dogs. Fur- 
ther work proceeds with the pure isomers. Thus, 



alert pursuit of a discrepancy led to a new endo- 
crinologic observation. 

Indirect Effects on Erythrocytes. The mecha- 
nisms of anemia in the leukemias and other can- 
cers have been reported previously by Dr. N. Ber- 
lin and colleagues. In the past year their atten- 
tion has turned to tumors and diseases associated 
with erythropoietin production. Dr. T. Wald- 
mann, in collaboration with Dr. Wendell Kosse, 
has found an increased mass and rate of synthe- 
sis of erythrocytes in patients with cerebellar 
hemangioblastoma, hypernephroma and pheo- 
chromocytoma. Non-dialyzable factors with 
marked erythropoiesis stimulating activity were 
isolated from the cystic hemangioblastoma and 
from the plasma and tumor homogenate of the 
patient with pheochromocytoma. Erythropoietin 
was also demonstrated in the urine and plasma 
of three patients with refractory anemia of un- 
known etiology. Autopsy in seven such patients 
revealed hemosiderosis of the liver, an organ 
which has been suggested as the site of erythro- 
poietin degradation. Dr. Waldmann found that 
dogs did not destroy erythropoietin during a 
single passage through the liver. He has also 
shown in ingenious experiments with parabiotic 
rats that the kidney is the major but not the sole 
site of erythropoietin production. Two other 
findings of interest in the dog were that cere- 
bellectomy caused no change in red cell mass but 
administration of desiccated thyroid was associ- 
ated with a 25 percent increase. 

Unexplained Indirect Effects of Cancer. One 
of the great enigmas of cancer is the mechanism 
by which the hot tissues are depleted during rapid 
tumor growth. In order to examine this, Dr. 
Berlin has developed techniques to determine 
gross body composition and total caloric ex- 
penditure. These methods have been applied to 
three markedly obese patients and nine with vari- 
ous neoplasms. He concluded that measurement 
of caloric expenditure in man, even with exten- 
sive tumor, is feasible but requires thirty days of 
observation on a metabolic balance regime. 
Agreement with caloric expenditure values cal- 
culated by the Newburgh insensible water loss 
method, was found in only one-half of the pa- 
tients. 
Dr. Bartter (NHI) and his colleagues have 



NATIONAL CANCER INSTITUTE 



11 



published accounts of a hemodilution syndrome 
with urinary salt wasting occurring in associa- 
tion with mediastinal tumors, possibly related 
to inappropriate secretion of antidiuretic hor- 
mone (ADH). Drs. Tschudy and E. Hellman 
have found a similar situation in the crises of 
acute hepatic porphyria, apparently related to 
the hypothalamic lesions of this disease. Dr. 
Tschudy's work on the biochemical lesion of 
porphyria has led to a finding of interest in ani- 
mal tumor pathogenesis. As noted previously, 
the livers of most tumor-bearing animals show a 
decrease in delta-aminolevulinic acid dehydrase, 
an enzyme on the pathway of porphyrin bio- 
synthesis. However, a possible exception is found 
in tumors due to polyoma virus, where no de- 
crease was observed until very late in the course 
of tumor growth. 

Dr. E. McLaughlin has found a marked dif- 
ference in the biological properties of serum of 
cancer patients as compared to normals. The 
method involves the effect of serum on the rate 
of mitotic activity in the regenerating livers of 
partially hepatectomized Sprague-Dawley rats. 
Dr. McLaughlin measured the livers for dry 
weight, mitotic counts and amount of tritiated 
thymidine incorporated into DNA. Serum from 
patients without cancer inhibited liver regenera- 
tion by all three measurements. By dry weight 
measurements, less than 50 percent of the liver 
regenerated. When serum from patients with 
cancer was injected, in excess of 70 percent of 
liver regenerated. This property does not fade 
when a primary tumor is excised or irradiated. 
Serum from animals with transplanted tumors 
inhibited regeneration, while that from animals 
with spontaneous tumors stimulated regeneration. 

Management of Cancer 

As pointed out previously, cancer in man by 
virtue of its ability to interfere with all bodily 
functions can induce physiologic infectional or 
emotional deficits in any combination. The ade- 
quate management of cancer must include their 
early recognition and correction. This report 
and previous reports have given many examples 
of the contributions of our clinical investigations 
to the further definition and control of such dis- 
tortions of the bodily economy. One instance 
will suffice to illustrate the role of research in 



providing rational medical support to the patient 
with cancer. Drs. Paul Schwab and Fahey have 
continued their work on Waldenstrom's macro- 
globulinemia. As noted last year, the macro- 
globulinemia is associated with a marked rise in 
plasma viscosity. Several patients have been 
treated by repeated plasmaphereses, with reduc- 
tion in macroglobulins and plasma viscosity. In 
addition, papilledema, retinal vessel dilitation 
and hemorrhages, and in one instance, congestive 
failure receded as the plasma viscosity was re- 
duced. 

Although these types of studies are an essential 
part of the rational management of the cancerous 
patient, they deal with remote effects and do not 
necessarily improve ability to control cancer. 
The modalities available to the clinician are the 
classical quartet of surgery, radiation, hormonal 
manipulation and chemotherapy. Endocrinologic 
aspects have been previously discussed; a few 
remarks follow on new information gathered in 
the other areas. 

Surgery 

Operative Effectiveness. Programs of curative 
surgery at NCI have been limited to relatively 
far advanced cancer of the head and neck, and of 
the uterine cervix. These studies are of sufficient 
size that it has been possible during the past year 
to obtain an estimate of the effectiveness of re- 
section in this unusual type of extensive but still 
localized cancer. 

Drs. Arons and Smith have analyzed the re- 
sults in 89 patients with cancers of the head and 
neck, admitted between July 1953 and January 
1960. All patients were followed through Au- 
gust 1, 1960, giving a range of followup periods 
of 6-76 months. Life table analyses were made 
of the 72 patients subjected to definitive surgery. 
At 2 years, 23 percent had clinical evidence of 
distant metastases, and at 3 years, 47 percent 
showed local recurrences. Forty percent survival 
at 60 months was calculated from the life tables. 

Dr. Robert Hoye studied the metastatic pat- 
tern in the 42 patients who were autopsied. 
Forty-five percent of these patients had disease 
limited to a potentially curable site. Fifty-five 
percent of the patients had metastases below the 
clavicles; the lung was involved in all but one, 
but this was apparent ante-mortem in less than 



12 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



half of the patients. This meticulous followup in 
both studies of head and neck cancer patients 
shows that although distant metastases are per- 
haps less common than with some other cancers, 
it could be demonstrated during life in one- 
fourth of the patients, and at autopsy in one- 
half. 

Drs. Smith and John Dillon similarly reviewed 
their experience with 131 patients with carcinoma 
of the cervix, admitted between August 1953 and 
January 1959. Only 120 of the patients were 
proven to have uterine cancer, and of these, 117 
were epidermoid cancer. Most of the cancers 
were relatively late; only three patients were 
classifiable as stage I. Management was as fol- 
lows: 

29 patients — clearly inoperable on admission 
31 patients — explored and found inoperable 
17 patients — radical hysterectomy and node 

dissection 
8 patients — anterior exenteration 
35 patients — total pelvic exenteration 
Operative mortality for the 60 patients in 
whom resection was attempted was 2.3 percent. 
As of May 1960, 9 of the 17 patients who had 
hysterectomy, and 17 of the 43 patients who had 
exenteration, were alive and free of tumor. Of 
the 34 patients who have died, 9 were free of 
tumor. In the 7 patients with hysterectomies and 
recurrence, 6 were local and 2 distant; in the 24 
patients with recurrence after exenteration, 12 
were local and 18 were distant. Dr. Smith con- 
cludes that the surgeon should do more radical 
surgery in some cases selected for hysterectomy, 
but avoid it when disease is clinically extensive. 
The two studies contain many valuable obser- 
vations on the natural history of these common 
cancers, have helped to define the place of radical 
surgery, and have resulted in the complete ac- 
ceptance of these procedures by the local medical 
community. 

Complications of Radical Surgery. Two of the 
circumstances which sharply limit the safety and 
completeness of surgery are wound infection and 
hemorrhage. Studies during the past year by 
physicians of the Surgery Branch have added 
vital facts and have provided the means of pre- 
vention. 



Wound Infections. Drs. Jack Bloch and 
Ketcham found 41 wound infections in 247 op- 
erations. Thirty of these were due to Staphylo- 
coccus aureus hemolyticus, coagulase positive, and 
20 of these occurred in ischemic tissues. Twenty- 
four of the 30 patients carried staphylococci pre- 
operatively, and where typing was possible, the 
types were the same — pre- and postoperatively. 
There was no evidence for cross-infections among 
patients, personnel or in the operating suite. A 
prospective study was then designed, with ran- 
domization and coding techniques, in which half 
the patients received Chloromycetin (4.0 grams 
daily) and half a placebo. There was a 14 per- 
cent occurrence of wound infection in 43 Chloro- 
mycetin treated patients, whereas 52 percent of 
the placebo treated patients had wound infec- 
tions. The investigators concluded that (1) 
wound infections in their patients occurred 
largely as a result of operating in areas which 
contain some ischemic tissue and are preopera- 
tively contaminated by staphylococci, and (2) 
under these conditions, prophylactic chemother- 
apy is required. 

OPERATrvE and PosTOPERATrvE Blood Loss. Al- 
though the need for replacement of blood at- 
tendant upon surgery is well understood, the use 
of the hematocrit as the measure of repair may 
lead to substantial under—- or over estimates of 
amounts required. Drs. Ketcham and Hoye, col- 
laborating with Dr. Berlin, have employed two 
techniques which allow for precise restoration. 
The first of these is the use of radioactive chro- 
mate labeled erythrocytes for the quantification 
of total red cell mass. Sixteen patients were 
studied pre- and post-operatively ; blood was re- 
placed as indicated by hematocrit. In three in- 
stances the total red cell mass was constant; in 
three patients there was a net gain of about 250 
ml. of red cells. In the other 10 patients there 
was a failure to replace blood varying up to 800 
ml. of red cells. In addition, an occasional pa- 
tient who had been transfused up to a normal 
preoperative hematocrit was substantially anemic 
in terms of red cell mass. In the postoperative 
period, there was a further decrease in total red 
cell mass, indicating a loss of 160 to 477 ml. of 
red cells over 14 days; a loss which could not 
be recognized grossly or by means of the hemato- 
crit. Thus, the repetitive study of red cell mass, 



NATIONAL CANCER INSTITUTE 



13 



can avoid the dangers related to too many or too 
few transfusions. 

The second technique employed in the operat- 
ing room has been the utilization of the LaVeen- 
Rubricus blood loss meter, which by an estima- 
tion of blood chloride permits a direct cumula- 
tive measure of operative blood loss. Not only 
did they collect and estimate blood lost by suc- 
tion, but also the blood on sponges, pads and 
towels. In nine patients there was a 370 to 2,700 
ml. deficit in unreplaced whole blood. In an 
additional 7 patients, they also measured the 
blood on drapes and gowns which ranged from 
350 to 1,465 ml. Further study during the 14 
days after surgery showed further reductions in 
red cell mass. The surgeons concluded that: (1) 
the hematocrit is an unreliable guide in estimat- 
ing operative and postoperative blood loss; (2) 
15 to 42 percent of operative blood loss is on the 
drapes and gowns ; (3) following radical surgery, 
the insidious loss of blood may be substantial, 
representing 9 to 44 percent of total red cell mass 
present immediately postoperatively. 

Radiation 

Radiation therapy, while curative of few histo- 
logic types of tumor, remains the principal means 
of providing prolonged regression of nonre- 
sectable cancer. Despite its pre-eminence in pal- 
liation, it has not been possible to expand seri- 
ously its curative potentiality by use of higher 
energies or better geometry. Tissue culture and 
animal evidence that chemical modification of 
the X-ray effect, or the use of other particles such 
as neutrons, give greater tumor damaging effects, 
has yet to be put to clinical trial. The program 
in the Radiation Branch during the past year 
has in the clinic continued to examine the useful- 
ness of the 2-million volt external X-ray beam in 
the management of cancer, either alone or with 
chemotherapy, and its effects upon bone marrow 
and renal function. In experimental animals, 
studies have centered about the physiological 
processes during recovery from radiation injury 
and the effect of X-rays upon immune response. 

Clinical Studies. Drs. J. R. Andrews and K. 
Brace have long been interested in time as a 
modifier of the radiation response. Clinical ra- 
diation therapy has classically been delivered 
during a 4-to-6-week period. Previous reports 



have shown that no advantage was apparent 
when the dose was given during a 100-day sched- 
ule. During the past year they have studied the 
effects at the other end of the scale, namely 2,500 
r given in a single dose of 40 minutes' duration. 
Preliminary observations indicate a good tumor 
response without complications. While it will 
be impracticable to make a definitive comparison 
with standard dosage schedules, it should be pos- 
sible to make some rough estimate of the effec- 
tiveness and safety of a single dose. If feasible, 
such a schedule would radically affect current 
practice of radiation therapy as well as permit a 
study of radiation as adjuvant on the day of 
surgical resection. 

Dr. Bruce has made several studies of the en- 
hancing effect of chemicals upon radiation re- 
sponsiveness. He has shown that Actinomycin D, 
which sensitizes cells in tissue culture to X-rays, 
similarly increased the radiation response of skin. 
In three carefully studied patients, however, there 
was no sensitizing effect of this antibiotic ' on 
tumors. Similar observations were made on the 
enhancing effects of HN 2 upon radiation therapy 
of bronchogenic cancer in a collaborative study 
with the Eastern Group. Disappointingly, nei- 
ther survival time nor tumor response was im- 
proved by the addition of the alkylating agent. 

Drs. J. R. Andrews, Brace, J. Levin and Berlin 
have studied the effects of total body irradiation 
on erythropoiesis. Patients studied had either 
disseminated lymphona or chronic lymphocytic 
leukemia. They received 85-100 r with 2.& MEV 
X-rays uniformly distributed to the entire body. 
The depression of peripheral counts was pro- 
found and reached a maximum for all elements 
at about 28 days after irradiation. The most 
sensitive index of bone marrow effect was the 
plasma iron disappearance rate. It showed maxi- 
mum depression at 2 days and a return to normal 
one week after irradiation. There was also a 
curious second fall at 28 days. The therapeutic 
responses obtained from total body irradiation 
were striking; — especially in the patients with 
chronic lymphocytic leukemia, where normal 
counts were still present 6 months after therapy. 

Drs. L. Avioli and Brace have undertaken a 
systematic study of the effects of abdominal X- 
irradiation upon renal clearances. Patients with 
large X-ray sensitive intra-abdominal tumors 
have been given 400-500 rad in single doses with- 



14 



ANNUAL EEVIEW OP INTRAMURAL RESEARCH 



out consistent change in glomerular nitration 
rate, renal plasma flow or TmPAH. 

Dr. Andrews and his colleagues have continued 
their studies of the use of S 3B in the treatment of 
chondrosarcoma. Two additional patients have 
been treated during the past year. It is clear 
that these cartilaginous tumors variably concen- 
trate the S 35 , presumably in chondroitin sulfate, 
and that the therapeutic utility is limited by 
bone marrow depression. Only one of the five 
patients is still in remission after administration 
of the radioisotope. 

Animal Studies. Dr. Roger Berry, who has re- 
cently joined the staff, has adapted a method de- 
veloped in England for in vivo examination of 
chemical enhancement. He used a lmphocytic 
leukemia in the ascites form in DBA/2 JN mice. 
Serial dilutions of the ascitic fluid were injected 
into groups of mice in sufficient number to ob- 
tain a 50 percent take value, thus permitting 
quantitative estimation of X-ray dose-response 
characteristics with and without chemical or 
physical adjuvants. A dose response curve for 
3 MEV X-rays has been established. If hydro- 
gen peroxide is given intraperitoneally prior to 
irradiation, a steeper dose response slope was ob- 
tained; the "oxygen enhancement" ratio approxi- 
mating 2.5 :1. In addition, Dr. Berry studied the 
combined effects of X-rays and 5-bromodeoxy- 
uridine or 5-iododeoxyuridine. Both combina- 
tions produced a marked decrease in the number 
of surviving cells compared to either modality 
used singly. Using the same system, with the 
kind cooperation of the staff of the Brookhaven 
National Laboratory, Dr. Berry has established a 
dose response curve for fission spectrum fast neu- 
trons. 

Dr. W. Smith has continued her survey of the 
processes involved in spontaneous and induced 
recovery from irradiation damage. She reports 
that (1) the time of spontaneous granulocyte re- 
covery varied with age in mice and correlated 
with ability to survive irradiation, and (2) in 
granulocyte mobilization studies, the ratio of 
mobilized to nonmobilized cells was similar for 
irradiated and normal mice. Endotoxin injec- 
tion, which improves survivorship, caused a four- 
fold increase in granulocytes. 



Dr. F. Smith has studied the effect of irradia- 
tion upon the ability of mice to form hemolysin 
to sheep erythrocytes. Irradiation diminished 
antibody production and this was a function of 
total dose rather than rate administration of X- 
rays. In rats, he found that altitude acclimatiza- 
tion increased capacity to form antibody hemoly- 
sin. Dr. Smith has also studied immune re- 
sponses in C 3 H mice with the plasma cell tumor 
X-5563, which were given X-irradiation and spe- 
cific antibody. Return of the immune response 
to near normal limits was observed in the tumor- 
bearing mice immunized as early as 24 hours after 
irradiation. 

Dr. C. Maxwell in his long-term studies of the 
effects of irradiation of amino acids has added a 
thirteenth compound, glycolic acid, to the list of 
materials appearing when solutions of glycine are 
irradiated with X-rays. Dr. P. Riesz has evi- 
dence for two kinds of hydrogen atoms differ- 
ing in chemical reactivity which appear in the 
abstraction reactions resulting from X-irradia- 
tion of water. Dr. H. Andrews found that mice 
can sense an X-ray beam under some circum- 
stances. This is probably related to ozone pro- 
duction. He has also developed dose response 
data for swine from the mid-lethal range to sev- 
eral thousand r. Offspring from heavily irra- 
diated parents had a dose response curve identical 
to those of the parents. 

Drs. M. Kelly, R. W. O'Gara and T. L. Loo 
have continued their studies of chemical protec- 
tion against X-rays and radiomimetic drugs. 
Previous studies have shown that amino-ethyl- 
isothiuronium (AET) protects against both and 
that those tissues which highly localize the drug 
receive the most protection. C14 labelled AET 
has been used to study its intracellular localiza- 
tion by microautoradiograms. It was found dis- 
tributed uniformly throughout the cytoplasm, 
without intranuclear incorporation. Compounds 
which generate free radicals have been found to 
be radioprotective. Di-t-butyl peroxide or t-butyl 
perbenzoate was injected intraperitoneally in 
mice or rats 30 minutes before HN 2 or whole 
body X-irradiation. These agents reduced the 
lethal effectiveness of HN 2 or X-ray by about 
50 percent. In studies with Dunning leukemia, 
neither drug interfered with the therapeutic ef- 
fectiveness of HN 2 . 



NATIONAL CANCER INSTITUTE 



15 



Chemo therapy 

General Remarks. Cancer chemotherapy has 
reached a new phase, and it is time to adjust at- 
titudes and planning to this perspective. From 
the observations of Dr. Hertz and his colleagues 
on trophoblastic tumors, it is clear that a drug 
can induce at least 5-year survival of patients 
with cancer. This means that a drug, in one 
type of cancer, not only assumes a place with 
surgery and radiation as primary therapy for 
cancer, but also can accomplish what surgery 
and radiation could never attempt, the control of 
diffuse metastatic disease. One problem then is 
the exploitation of this success, to identify the 
screening system which could have predicted it. 
This concept should be extended to the whole 
search for chemical control of cancer — namely, 
the task of proving the value of a biological sys- 
tem which can predict drug responsiveness for 
each of the many diseases known as cancer. Five 
years ago there was confusion in drug screening 
because of the number of variables and the lack 
of quantitative data for either man or animal. 
Due to the efforts of CCNSC, the cooperation of 
scientists throughout the country, and I believe 
the leadership of several NCI scientists and phy- 
sicians, quantitative data have been gathered and 
the beginnings of such screening correlates are 
within our grasp. Predictability seems highly 
probable for acute leukemia, possible for chorio- 
carcinoma, and other screens, both transplanted 
animal tumor and tissue culture, are partly cate- 
gorized for the compounds they find or miss. 
Finally, the easy convenience of transplanted 
rodent tumors has been in part abandoned for 
exploration of the more difficult screening tech- 
niques of spontaneous tumors and those induced 
by viruses, carcinogens and radiation. For the 
above reasons, it is possible to abandon in large 
part any random search for the panacea, and to 
focus on particular cancers. This means that the 
start must be made with man and that the basis 
for establishment of screening specificity should 
be the chemicals which control specific cancers in 
man. 

The Intramural Chemotherapy Program or 
NCI. The intramural program has been devel- 
oped on the assumption that it was an integral 
part of the national effort in cancer chemo- 



therapy, and yet had resources and interests of 
its own. It was felt that thus our scientists 
could actively share work load and data with 
others similarly motivated, and still be able to 
pursue imaginatively their own research ideas. 
Since it was undesirable to duplicate expensive 
facilities, the intramural group developed no syn- 
thetic program or primary screen of its own, but 
depended entirely upon CCNSC. As its share, 
the intramural program pioneered with quantita- 
tive screening, developed data on toxicity and 
pharmacology of new agents and was responsible 
for the leadership of two of the clinical groups. 
The ground to be covered was so vast, the ob- 
jectives so uncertain, that several years of trial 
and error were required to develop rational 
methodology. During the past 5 years many of 
these methodologic puzzles have been solved, and 
the intramural program is ready to plan the next 
phase, that of utilization of calibrated techniques 
for the uncovering of chemical control of some 
specific cancers. 

Specific Anti-Tumor Agents. (1) Anti folic 
Compounds: Drugs with antifolic activity were 
among the first antitumor agents to be discov- 
ered. Synthesis of these compounds was extraor- 
dinarily difficult, and after the first few com- 
pounds failed to show increased activity in micro- 
biological systems, chemists turned with relief to 
the newest drug, 6-mercaptopurine. Despite the 
ease of synthesis of the antipurines, 6-mercapto- 
purine has proved of narrow utility. Metho- 
trexate, an antifolic, not only induces complete 
remission in acute lymphocytic leukemia and 
metastatic trophoblastic tumors, but has been 
shown by Dr. Van Scott to cause regression of 
basal cell cancer, and by Dr. Frei and his col- 
leagues to cause regression in several lymphomas. 
NCI, thanks to CCNSC and intramural joint 
efforts, has been able to undertake a broad struc- 
ture-activity synthesis program with Southern 
Research Institute. 

One of the reasons for the interest in antifolic 
synthesis, has been the discovery by Dr. Goldin 
and colleagues of the increased activity of anti- 
folics brought about by halogenation in positions 
5 and 6. As previously reported, he found in 
advanced L1210 leukemia in mice that dichloro- 
methotrexate or bromochloro-methotrexate as 
compared to methotrexate, effected a threefold 



16 



Activity 100 

in 
Childhood 
Leukemia 
(Mtx= 
1007.) 

50 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 
COMPARISON OP THERAPEUTIC INDICES 2 

CHILDHOOD LEUKEMIA vs. MOUSE L1210 



MP 



Mtx 



DCM 



Pr 



ITG 



GA 



Cyt 



* JV A Sa Ac FU 
50 



100 



150 



200 



250 



Activity in L1210 (Mtx = 100%) 



*Azauracil, Nitrosoguanidine, Narcotine, 

V=Vincaleucoblastin, A=Actinomycin D, Sa=Sarcolysin, A=Azauridine, 
FU=Flourouracil, Pr= Prednisone, MP =Mercapto purine, 
Mtx = Methotrexate, ITG = Imidazolylthioguanine, GA = Guanylhydrazone, 
Cy t = Cytoxan, DCM = Dlchloromethotrexate 



increase in survival time. This quantitative 
screen has developed into a predictability system 
of considerable value for acute lymphocytic leu- 
kemia as shown in the accompanying chart de- 
veloped by Drs. Frei and Goldin : 

As a working hypothesis, those compounds with 
greater than 50 percent of the activity of metho- 
trexate can be expected to be active in acute 
lymphocytic leukemia. It has been disappoint- 
ing to recognize that the most active compounds 
yet found, the halogenated antifolics, have not 
been similarly more effective in leukemia. Dr. 
Frei and colleagues, working with Leukemia 
Group B, found that oral dichloromethotrexate 
was no more active than methotrexate in acute 
lymphocytic leukemia. The reason for this was 
thought located when Dr. Goldin reported that in 
L1210 oral administration of dichloromethotrex- 



2 The rational use of animal screens as predictability systems 
for cancer in man requires quantitative data such as these. It 
has taken 5 years of intensive effort to collect data for this 
figure. Note that it involves one animal tumor and a single 
disease. A similar figure cannot at this time be constructed for 
any other cancer in man. The magnitude of finding curative 
drugs for several cancers in man should not be underestimated. 



ate was associated with sharp loss in therapeutic 
effectiveness. Repetition of the clinical study by 
Leukemia Group B, using parenteral drug, did 
not appear to increase activity in acute lympho- 
cytic leukemia. Intensive studies of the pharma- 
cology of dichloromethotrexate by Drs. Oliverio, 
T. L. Loo, J. D. Davidson, D. Kail, and Mr. R. 
Dion, have provided a clue though perhaps not 
the full solution, to the discrepancy By the use 
of DEAE ion-exchange columns, they were able 
to separate and qualify various folic acid ana- 
logues and found that 40-50 percent of dichloro- 
methotrexate was excreted in the urine in the 24 
hours following a parenteral dose. Moreover, 
one-fifth of the dichloromethotrexate was in the 
form of a metabolite, characterized as the 4:7- 
dihydroxy derivative. They also synthesized di- 
chloromethotrexate containing radioactive chlo- 
rine, and found that it was rapidly excreted in 
the bile of rats, mice, dogs and man. Since it 
is poorly absorbed from the intestine, biliary ex- 
cretion effectively removes the drug from the 
blood. The parent compound because of these 



NATIONAL CANCER INSTITUTE 



17 



two processes rapidly lost its effectiveness. This 
does not explain the discrepancy with the screen- 
ing animal, since qualitatively at least the phar- 
macological differences between mouse and man 
are not great. 

Drs. Hertz, Lipsett, Ross and colleagues have 
now treated 68 women suffering from trophoblas- 
tic disease with antifolics. Forty-five percent of 
the patients are in complete remission after 
methotrexate treatment. The first few patients 
treated have sustained their remissions for over 

5 years. The possibility that the therapeutic re- 
sponse is related to maternal immunization by 
fetal tissues cannot be supported. In the first 
place, methotrexate seriously interferes with 
many immune phenomena. Secondly, Dr. Hertz 
in a study of red cells and plasma from 35 
couples in the above series, could find no evidence 
of maternal immunization. The patients who 
have relapsed have been refractory to nitrogen 
mustard, Cytoxan and actinomycin D. However, 

6 of 14 methotrexate-resistant patients have re- 
sponded to an alkaloid, vincaleukoblastine. Two 
of these have remained in complete remission for 
8 and 10 months respectively. The manner in 
which this compound was selected is highly per- 
tinent to the previous discussion of screening. 
Dr. Hertz has been able to establish heterologous 
growth of human choriocarcinoma in the cheek 
pouch to the untreated hamster in 6 of 35 at- 
tempts. Some of these were established prior to 
treatment and are methotrexate-sensitive. Others 
are methotrexate-resistant and are used to screen 
potential antitrophoblastic drugs. Two active 
compounds have been found, vincaleukoblastine 
and podophyllotoxin, and it is of interest that 
neither has activity in the L1210 screen or in 
acute lymphocytic leukemia. Thus these two 
methotrexate-sensitive tumors — trophoblastic dis- 
ease and acute lymphocytic leukemia will likely 
have entirely different screens for predicting ac- 
tive drugs. 

(2) Purines and pyrimidine antagonists: The 
discovery that 6-mercaptopurine was an effective 
drug in the leukemias brought a new measure of 
hope for the chemical control of these diseases. 
Not only was 6-mercaptopurine less toxic than 
the antifolics, it had activity in the myelocytic 
leukemias, both acute and chronic, as well as in 
the acute lymphocytic form. Dr. Frei and his 
colleagues in collaboration with Leukemia Group 



B have completed their definitive comparisons of 
6-mercaptopurine and methotrexate, as reported 
last year. They have gone forward in their 
methodologic studies to examine the usefulness of 
sequential design in 6-mercaptopurine treated 
acute leukemias. All previously untreated chil- 
dren with either type of acute leukemia were 
given prednisone. When remission occurred the 
patients were placed on continuing 6-mercapto- 
purine or placebo. The duration of remission 
was used as the measure of effect and it was 
quickly shown that a small number of patients 
could demonstrate the active drug. Moreover 
this design, since it incorporates initial therapy 
with steroids, permits the use of new drugs in 
the study of untreated patients. The safety of 
this maneuver is further reinforced by the fact 
that relapse after steroids plus new drug can be 
fully treated with 6-mercaptopurine or methotrex- 
ate. Finally, it has the further advantage that 
new drugs can be given while the patients are in 
steroid remission, and there is little danger of 
confusing toxicity with the complications of acute 
leukemia. 

The screen which is best for indicating anti- 
purine type of activity is CA 755. Dr. Goldin 
and his colleagues have developed this system 
into a reliable quantitative measure of compara- 
tive activity of thio-purines. The only com- 
pound of greater activity than 6-mercaptOpurine 
has been its riboside. Preliminary studies in 
man indicate little advantage, but the definitive 
comparison is in the future. Imidazolyl thio- 
guinine, which is somewhat less effective against 
755, had less activity than 6-mercaptopurine in 
52 patients with acute leukemia treated by Leu- 
kemia Group B. Recently, CCNSC held a con- 
ference about the thiopurines in which Drs. 
Goldin and Frei participated. There was gen- 
eral agreement to limit the synthesis of these 
compounds until 6-mercaptopurine riboside ac- 
tivity in man is fully explored, and more is 
known about the effect of 9-ethyl, and 9-butyl-6- 
mercaptopurine in man. 

In spite of the failure of the synthesis efforts 
to produce better drugs, 6-mercaptopurine has 
provided an excellent model for biochemical 
studies of resistance. Dr. Davidson has previa 
ously reported studies of L1210 leukemia in which 
resistant leukemic cells showed reduction in ino- 
sinic phosphorylase. Studies in the past year 



18 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



with the isolated enzyme have confirmed the cel- 
lular findings of competition between 6-mercap- 
topurine and hypoxanthine for inosinic phos- 
phorylase. Dr. Davidson also has developed new 
techniques for the assay of the enzyme in human 
leukemic cells. Eighteen patients have been 
studied and all showed some ability to convert 
hypoxanthine C u and 6-mercaptopurine S35 to 
nucleotides. Four apparently 6-mercaptopurine 
resistant patients have been studied and two of 
these had normal amounts of the enzyme, sug- 
gesting that their resistance was not the same as 
that of mouse leukemia L1210. 

Other studies of leukemic leucocytes with a 
pyrimidine antagonist, azauridine, have been car- 
ried out by Dr. H. Fallon. Azauridine is known 
to inhibit orotidylic decarboxylase, essential for 
pyrimidine biosynthesis. Dr. Fallon has found 
that the enzyme in leukemic leucocytes is pro- 
foundly inhibited by drug. Azauridine causes a 
sharp fall in the peripheral leucocyte count of 
leukemic patients and this correlates well with 
enzyme inhibition. 

Finally, intrathecal 6-mercaptopurine riboside 
is being used for the treatment of leukemia in- 
volving the central nervous system. As reported 
last year, central nervous system leukemia is 
occurring with high frequency and is a common 
cause of treatment failure. This is largely be- 
cause of failure of methotrexate or 6-mercapto- 
purine to achieve therapeutic concentration in the 
central nervous system. Dr. Rail and colleagues 
have continued their studies of the principles of 
drug transfer from blood to the central nervous 
system. Working both with dogs and dogfish, 
they have evidence that the failure of acids, such 
as sulfanilic and para-amino hippuric, to achieve 
high concentrations, in spite of continuous high 
blood concentrations, is due to bulk out-flow of 
cerebrospinal fluid (CSF), removing fluid and 
drug at such a rate that inward transfer rates are 
inadequate to reach a ratio of unity. Drs. Rail 
and Streicher (NIMH) have for the first time 
found a compound, thiocyanate, which has a 
variable CSF/plasma ratio related to plasma con- 
centrations. At plasma thiocyanate concentrations 
of 8-10 mM/L, the ratio approaches unity, while 
at 1-2 mM/L the ratio is 0.1. They suggest that 
this is evidence that the bulk return of CSF into 
blood is an active process. 

Binding to proteins limits passage of drugs into 



the CSF. Dr. Rail and his colleagues, in study- 
ing the protein binding in various species, have 
noted the absence of albumin in the dogfish with 
predictable effects on central nervous system 
transfer of drugs. They also report the absence 
of albumin from the plasma of sea lamprey larvae 
(P. marinus dosatux) . Associated with metamor- 
phosis into the adult, there appears a new plasma 
protein, with electrophoretic characteristics dif- 
ferent from mammalian albumin. 

(3) Alkylating agents: The first non-hormonal 
drug shown to affect disseminated cancer was 
nitrogen mustard. It clearly can cause substan- 
tial regression of Hodgkin's disease and other 
lymphomas, both forms of chronic leukemia, my- 
cosis fungoides, neuroblastoma and adenocarci- 
noma of the ovary. It has erratic and insubstan- 
tial effects in other epithelial tumors and none 
in the acute leukemias. In the 15 years since the 
discovery of nitrogen mustard, many other alkyl- 
ating agents have been synthesized, shown to have 
activity in a variety of transplanted tumors and 
a few put into clinical trial. The programs of 
NCI, both of CCNSC and the intramural area, 
have undertaken considerable research concerning 
alklyating agents. Both groups have participated 
in recent conferences attempting to interpret the 
vast amounts of information. It is clear that 
alkylating agents vary considerably in quantita- 
tive and qualitative effectiveness in transplanted 
animal tumors. There is some evidence that this 
is also true in man — for example: (1) Myleran 
has much greater activity in chronic myelocytic 
leukemia than in any other disease; (2) Cytoxan 
has a modest degree of activity in acute leukemia, 
whereas the other alkylating agents do not; (3) 
Cytoxan has relatively little effect on megakaryo- 
cytes and platelets as compared to other alkyl- 
ating agents. Nevertheless the degree of differ- 
ence found in transplanted animal tumors is not 
generally reflected in man. I believe the difficulty 
may represent in an outstanding way what has 
been said above about screening. The guiding 
principle of the past has been to find a generally 
active drug in animal tumors which will be gener- 
ally active in human cancer. What is needed, be- 
fore the lead of the alkylating agents is aban- 
doned, is specificity — particular animals systems 
which can dependenably predict activity in a 
single human cancer. We have nowhere in alkyl- 
ating agent research approached the knowledge 



NATIONAL CANCER INSTITUTE 



19 



we have about L1210 and acute lymphocytic leu- 
kemia and Ca 755 and myelocytic leukemia. 

NCI intramural scientists are participating in 
several efforts to find such specificity, using trans- 
planted rodent tumors and induced tumors. With 
the former, Dr. Goldin has attempted to develop 
Sarcoma 37 into a quantitative screen. This 
tumor has been unaffected by all the standard 
drugs, and it was hoped that it would be an effec- 
tive screen for alkylating agents of high potency. 
Recently Cytoxan and alanine mustard have been 
shown to be active, but the meaning of this in the 
clinic has yet to be determined. 

Dr. Leon Schmidt of Cincinnati has undertaken 
a thorough study of the value of rat transplanted 
tumors in predicting drugs for specific cancers. 
He has found that relatively slight alterations in 
experimental design can change markedly the 
relative activity of an alkylating agent. With 
control over these experimental variables, he is 
now able to rank various major alkylating agents 
for order of effectiveness and is cooperating with 
clinicians and CCNSC in trying to relate these 
to clinical predictability. Because of the mass of 
data, this will probably be solved only with the 
help of computers. Dr. A. Pratt of the Physiol- 
ogy Laboratory and several NCI clinicians have 
worked with Dr. Schmidt in developing mathe- 
matical models suitable for the examination of 
this problem. When more clinical information 
is available on newer alkylating agents, the spe- 
cificity (or the lack of it) of these rat systems 
can be understood for the first time. 

Since it is possible that transplanted animal 
tumors may not provide the required specificity, 
nontransplanted tumor systems are being devel- 
oped. Dr. Goldin and his colleagues have de- 
veloped the Moloney leukemia virus into a par- 
tially quantitative system. They have also been 
attempting to use similarly the spontaneous breast 
tumors arising in the mouse colony at NLH. In 
both instances preliminary results with drugs 
warrant the development of larger supplies of 
these tumor types. 

Drs. M. Kelly and R. O'Gara have been study- 
ing drug effects in carcinogen-induced tumors. 
Chemical carcinogens were given to new-born 
mice; they developed pulmonary tumors, leu- 
kemia and fibrosarcomas. Drug therapy was 
given from age of 8 weeks to 16 weeks, and the 
frequency of pulmonary tumor nodules noted. 



In preliminary observations alkylating agents or 
cortisone had more effect than antimetabolites. 

o, //-DDD (2,2-bis (2-chlorophenyl-4-chloro- 
phenyl) -1,1-dichloroethane) 

Dr. Hertz and his colleagues have continued 
their studies of the effect of this adrenal cortical 
inhibitor upon adrenal cortical cancer. Eighteen 
patients have been treated and all but 4 have 
shown depression of tumor hormone output. 
Seven patients have had regression of metastatic 
disease and clinical improvement. Damage to 
normal human adrenals has also been demon- 
strated. Pharmacologic studies have shown that 
much of the drug appears in the stool after oral 
administration. Nevertheless infusions of the 
drug in Lipomul did not increase activity. The 
drug is concentrated in fat depots which rapidly 
remove circulating compound. 

This compound is of interest because it was 
discovered without the aid of an animal tumor 
screen, since to date it has not caused regression 
of animal tumors. Since it does not affect normal 
adrenal activity of rodents, "screening" for ac- 
tivity of analogues is carried out in the dog, 
whose normal adrenal cortex is highly sensitive. 
Dr. W. Tullner has found that duration of treat- 
ment and drug dose of o,p' DDD was correlated 
with degree of cellular destruction in the male 
dog. He prepared animals in which damage was 
confined to zona reticularis and zona fasciculata 
with the zona glomerulosa appearing normal. 
After cessation of drug, the adrenal secretion of 
17-hydroxysteroids was reduced to extremely low 
levels. Gradual recovery of hormonal output and 
normal histology appeared in 4 weeks and autopsy 
showed regeneration of zona fasciculata. Dr. 
Tullner concludes that the zona glomerulosa is 
multipotential and even though it produces al- 
dosterone and does not contain the enzymes for 
hydrocortisone production, it can give rise to 
fasciculata cells which can carry out this hy- 
droxylation. He has also shown that intravenous 
difluoro, dinitro DDD was as active in the dog 
as o,p' DDD, but possessed no advantage over 
that compound. 

Conclusion 

In conclusion, I shall indicate several areas 
where resources not now extant are required. 
Additional laboratory space in the Clinical Cen- 



20 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



ter is clearly inferred from the need to have 
deeper staffing. An "Operations Research" group 
is required to consider in a formal way clinical 
data management — its collection, coding, analysis 
— and the development of mathematical models 
for computational examination. Several areas 
are in need of primate breeding facilities for 
carcinogenesis studies in the new-born as well as 
for trophoblastic tumor pathogenesis. Resources 
are in demand for large mouse colonies for spon- 
taneous tumors and for special isolation facilities 
for chemotherapy and other studies of virus- 
induced tumors. Personnel and facilities should 
be planned for a systematic examination of pre- 
dictability value of animal systems for toxicity 
of drugs for man. In this regard electron micro- 
scopy of drug damaged structures will certainly 
be needed as light microscopy is non-specific for 
such pathology. Finally, personnel and resources 
should be planned for the expansion of clinical 
cytogenetics. 

I should like to extend to the Director, NCI, 
and Director, Clinical Center, and their staffs my 
gratitude for the kind and effective manner in 
which they have met the mercurial demands of 
the clinical program. May I also express my 
appreciation to Dr. Stuart Sessoms and his staff 
for their extraordinary help in working with us 
in the chemotherapy program. I want to make 
known my thanks to the Laboratory Chiefs and 
their staffs for their kindly help to the clinicians. 
I am grateful to Mr. Walter Magruder, Mrs. 
Martha Rees and the secretaries of OADR for 
their cheerful help in carrying out the functions 
of my office. Finally, it is a pleasure to acknowl- 
edge my indebtedness and thanks to the Clinical 
Branch Chiefs and all their physicians and to 
Miss Burgess and Mrs. Kilian and their staffs for 
courteous support in many ways, but especially 
in bringing to NCI patients the superb medical 
care which is the beginning of all clinical inves- 
tigation. 



NONCLINICAL INVESTIGATIONS 3 

Members of the staff who participate in the 
nonclinical programs continue to create new 
knowledge that enhances our understanding of 

3 Prepared by Carl G. Baker, M.D., Assistant Director (for 
Research ) . 



cancer, entails development of new concepts, and 
provides a reservoir of laboratory findings im- 
portant for future application to the problems of 
cancer and other diseases. The joint presence of 
this group of research workers and the clinical 
investigators within one sizable Institute provides 
the means for an exchange highly beneficial to 
both groups. The function of the nonclinical 
intramural program may be viewed as the crea- 
tion of new knowledge that is well ahead of that 
required for application to problems of man, and 
the yields of the program form a wellspring 
which must be maintained with a vigor that in- 
sures a wholesome level. Among the most im- 
portant ingredients is the continual supply of 
new concepts since they are the key to bringing 
new approaches to bear on the cancer problem 
beyond those receiving current consideration. 
Another important ingredient is the development 
of concepts already at hand to a stage sufficient 
for adequate evaluation. Collaboration by inves- 
tigators in different disciplines, founded upon 
mutual interest, understanding, and respect, can 
do much to bring about new concepts and to speed 
along their development. In a number of cases, 
sizeable groups, ably led, provide greatest prog- 
ress. In other cases, the mature investigator 
working independently makes his greatest con- 
tribution. Oftentimes the investigators who cre- 
ate new concepts and make initial development 
of them can make their best contribution when 
they do not carry through the application of the 
findings, but rather are left free to develop new 
concepts, understanding and evaluation from the 
purely scientific standpoint. In other cases, the 
investigator who initiates a study may effectively 
carry it through to a point where its applica- 
bility to an evident problem is attained. 

The summarization which follows of the re- 
search progress of the nonclinical intramural staff 
attests the continuing highly important contribu- 
tions made by the Institute staff. While all areas 
of cancer research are not fully represented, the 
staff is producing research of a very high order 
in many areas believed to be of significance for 
the future development of cancer research. 

Studies on the Induction of Cancer 

Several investigators throughout the Institute 
continue to make contributions toward our under- 



NATIONAL CANCER INSTITUTE 



21 



standing of the factors underlying the causation 
of cancer. Many aspects of this problem warrant 
increased effort, both in terms of the materials 
in the environment to which man is exposed, 
singly or in combination, and the mechanisms 
within the living organism that are involved in 
initiation of the cancer process. 

Chemicals 

Studies progress on the organic fractions of 
the particulate phase of urban air pollutants, as 
part of a comprehensive and prolonged program 
dealing with the relationships between air pollu- 
tants and health hazards, by Drs. W. C. Hueper 
and W. W. Payne in collaboration with Drs. P. 
Kotin and H. Falk (University of Southern Cali- 
fornia) and Messrs. E. Tabor and E. Sawicki 
(Taft Sanitary Engineering Center). Experi- 
ments with mice receiving the powerful carcino- 
gen, benzo[a]pyrene, which serve as a reference 
standard for comparing relative potency of the 
solvent fractions of air pollutants, confirmed the 
concept that a low-level recurring exposure to a 
carcinogen is more hazardous than a single expo- 
sure to the same amount. In addition to con- 
firming the presence of benzo[a]pyrene in samples 
collected by filtration of air from eight large 
United States cities, the group has also shown 
carcinogenicity of samples not containing benzo 
[a]pyrene. Distinct variations in composition of 
air pollutants from different cities were demon- 
starated in terms of biological and chemical 
parameters. The percentage of tumors observed 
differed with the chemical fraction studied as 
well as the city from which the sample was ob- 
tained. No consistent correlation between the 
degree of carcinogenic potency, the amount of 
benzo[a]pyrene per sample, and the lung cancer 
mortality rate reported for a given city was 
found. 

Preliminary studies on testing eluates of carbon 
adsorbates from raw river waters for carcino- 
genicity have produced several cancers in mice 
receiving samples subcutaneously. 

Drs. Hueper and Payne have extended the 
studies on carcinogenicity of chromium-contain- 
ing materials. Chromite ore roast, which con- 
tains various compounds of chromium with dif- 
ferent solubilities, produces tumors in rats, and 
the list of chromium compounds shown to be 
carcinogenic has been extended, with the addition 



now of a trivalent chromium compound along 
with other hexavalent species. Moderately solu- 
ble (water) hexavalent chromium compounds 
display the highest carcinogenicity. 

Prolonged inhalation by rats of dusts of 
chromic oxide and the chromates of barium, 
lead and zinc, compounds widely used as pig- 
ments and hence in contact with many workers, 
has produced green deposits, adenomatous 
changes and squamous cell metaplasia in the 
lungs of the exposed animals, but no primary 
cancers. 

Because tumor-inducing effects of certain-water 
insoluble polymers have been interpreted by some 
investigators as resulting from physical factors, 
e.g., surface forces, rather than chemical ones, 
Dr. Hueper investigated the tumor-producing 
abilities of chemically different polymers of this 
type implanted subcutaneously or intraperito- 
neally in rats and mice in different physical 
forms, such as cube, sheet, disk, film foam, and 
powder. Although data obtained with polyethyl- 
ene are consistent with the interpretation that 
physical factors play the key role in carcinogene- 
sis with polymers, the data obtained with poly- 
urethane are against the concept. 

Because previous testing of polyvinyl pyrolli- 
dones (PVP) containing a wide range of molecu- 
lar weights gave inconsistent results as to their 
carcinogenic properties and the variability of the 
findings might have been due to differences in 
retention of some of the larger polymer species, 
Dr. Hueper re-evaluated the carcinogenic prop- 
erties of PVP preparations containing either 
small (average molecular weight of about 10,000) 
or large (average molecular weight of about 
50,000) molecular species. Although deposits of 
homogeneously staining material were seen in 
various tissues in the rats and rabbits injected 
with PVP of both large and small molecular 
species, the cancers that appeared were the same 
as those seen in the controls (frequency, location 
and morphological types) . The smaller molecular 
weight PVP molecules were retained by the 
glomerular filter in the kidneys of the rat, but 
not in those of the rabbit. 

Several investigators have produced cancers of 
the urinary bladder in mice by surgically im- 
planting pellets of paraffin wax or cholesterol 
containing a carcinogen into the bladder by a 
method developed by Jull. However, bladder 



22 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tumors have also appeared in the control animals 
with pellets of paraffin wax or cholesterol alone. 
Drs. W. Conway, W. Hueper and E. Feldstein 
and Miss E. Lethco have, therefore, prepared 
pellets made of synthetic wax (hexadecyl palmi- 
tate) and have incorporated into the pellets in 
addition to the dyes under test, varying amounts 
of powdered cellulose to provide greater perme- 
ability of fluids into the pellets. They have 
shown that more rapid release of the test sub- 
stance from the pellet occurs with higher pro- 
portions of cellulose, thus providing the means 
for controlled release of the potential carcinogen 
under study. With this new technique for evalu- 
ating substances for carcinogenicity, several azo 
dyes are under study. 

Dr. Conway and Miss Lethco are also investi- 
gating the metabolism of several azo dyes by 
other techniques. Utilizing sensitive colorimetric 
and radioactive tracer techniques, they have ob- 
served several colored and colorless metabolic 
products in the urine. The percentage of the 
dose which appears as urinary metabolites in- 
creases as the dose decreases. 

Drs. M. G. Kelly and E. W. O'Gara have 
added to their observations on chemical carcino- 
genesis in newborn mice. After implantation of 
either 0.02 mg. dibenz(a, b) anthracene (DBA) or 
0.011 mg. 3-methylcholanthrene (MC) into non- 
inbred albino mice during the first 18 hours after 
birth, 100 percent of the animals developed pul- 
monary tumors by 16-29 weeks. Subcutaneous 
fibrosarcomas arising from perimysium appeared 
at the site of injection of either carcinogen. Mice 
given 3-methylcholanthrene developed fulminat- 
ing leukemia 9-16 weeks after injection. In col- 
laboration with Mr. Nathan Mantel, the dose 
response relations were studied. The smallest 
single doses producing tumors are: Fibrosarco- 
mas— 0.0002 mg. DBA and 0.0004 mg. MC; lung 
tumors— 0.0067 mg. DBA and 0.0037 mg. MC. 
At high doses of MC, 30 percent of albino mice 
developed leukemia and 70 percent of C3H male 
mice developed hepatomas. The production of 
tumors with such small doses suggests the value 
of the newborn animal as a test subject for poten- 
tial carcinogens of greater sensitivity than that 
available formerly. 

In studies aimed at inducing lung cancers, Dr. 
M. Stanton and Messrs. E. Blackwell and J. Al- 
brecht have produced metastasizing squamous-cell 



carcinomas of the lung in rats by intravenous 
injection of a suspension of methylcholanthrene 
in tricarprylin carried to the lung in an halogen- 
ated aliphatic hydrocarbon (hexachlorotetraflu- 
orobutante). The tumors apparently arose in 
areas of bronchial metaplasia, probably arising 
from halocarbon-produced infarction. Ultravio- 
let light microscopy indicated deposition of 
methylcholanthrene at the tumor site. 

Dr. E. W. Chu, with Dr. K. M. Herrold, is 
engaged in a study of the effects of carcinogens 
on the female genital tract of Syrian hamsters. 
Hamster cervices painted with 9, 10-dimethyl: 
1, 2-benzanthracene showed atypical cytological 
changes in the vaginal smears after iy 2 months, 
and definite carcinomatous changes in 2 months. 
3, 4-Benzpyrene-painted cervices showed compa- 
rable cytohistological findings after a much more 
prolonged period. Cytological changes have been 
noted in vaginal smears obtained from animals 
whose cervices were painted with tobacco tar dis- 
solved in acetone only after 10 months. 

Of 400 hamsters that received pellets contain- 
ing suspected carcinogens surgically implanted 
into the cheek pouch, Dr. L. Dunham found that 
24 animals displayed lesions in the pouch, of 
which 19 were inflammatory in nature. One 
squamous papilloma was seen and four examples 
of epithelial hyperplasia were noted. 

Further work on the hemangioendotheliomas 
of liver and spleen that developed following in' 
travenously injected Thorotrast (colloidal tho- 
rium dioxide) has been performed by Dr. E. 
Swarm. Metastases to spleen and liver oocur fol- 
lowing subcutaneous transplantation. Dr. P. 
Mori-Chavez has found visceral metastases with 
these tumors more frequent at high altitudes (in 
Peru) than at sea level. Dr. Swarm has also re- 
viewed in several European laboratories tissue 
specimens from human patients who were given 
thorotrast. 

Dr. M. Potter is investigating mouse strain re- 
lationships and the induction of leukemia by 
skin painting with methylcholanthrene. Approxi- 
mately 80 percent of one-month-old DBAV2 
mice painted with the carcinogen developed gen- 
eralized leukemia. No leukemias appeared in 
strain BALB/c mice which had been painted in 
a similar way. The Fi hybrids of BALB/c and 
DBA/2 also did not develop leukemia after skin 
painting. The animals of the first generation 



NATIONAL CANCER INSTITUTE 



23 



backcross to DBAV2) however, had an incidence 
of about 25 percent leukemia. Studies are being 
extended to a family study of second generation 
backcross mice. 

Mechanisms of Carcinogenesis 

Dr. H. Sidransky is investigating a number of 
factors influencing the pathogenesis of rat hepa- 
tomas induced by certain chemical carcinogens. 
Although liver regeneration in partially hepa- 
tectomized rats is somewhat inhibited in rats in- 
gesting ethionine in comparison with rats on the 
control diet, the histologic changes in the liver 
of the experimental animals are similar to those 
Seen in nonhepatectomized animals ingesting 
ethionine. However, rats with partial hepatec- 
tomies and ingesting ethionine appear to have a 
delayed induction period for hepatomas. p-Hy- 
droxypropiophenone, which inhibits the develop- 
ment of liver tumors induced by butter yellow, 
was found to inhibit ethionine-induced hepa- 
tomas. With Dr. H. Morris, Dr. Sidransky 
found that male rats show a higher incidence of 
liver tumors when fed N-2-fluorenylacetamide 
than female rats. If the survival of female rats 
is prolonged by removing breast tumors, the inci- 
dence of hepatomas can be increased appreciably 
in the female animals. 

Dr. Sidransky, with Drs. E. Farber (Tulane 
University) , M. Rechcigl and T. Baba, is investi- 
gating the effects of nutritional deficiencies on 
the pathogenesis of hepatomas in rats in an ef- 
fort to learn if other nutritional deficiencies be- 
sides chronic choline deficiency can lead to liver 
cancer. In short-term experiments, young rats 
were found to develop signs of acute choline de- 
ficiency only when the diet contained no choline 
and 0.12 percent or less of methionine. In long- 
term experiments, no signs of cirrhosis or hepa- 
tomas have yet appeared in rats on an adequate 
choline but methionine deficient diet for 22 
months. Species differences have been observed 
in determinations of liver choline oxidase ac- 
tivity ; among nine species, the rat has the high- 
est activity and human the lowest. Pathologic 
changes in the liver of the rat similar to those 
seen in kwashiorkor were observed within a few 
days after tube-feeding of young rats on certain 
essential amino acid deficient diets. 

Dr. H. P. Morris, with Mrs. B. P. Wagner, 
continues to add to the knowledge of the car- 



cinogenic activity of certain chemical compounds, 
structural and metabolic relationships, species 
differences involved, and dietary effects. Last 
year, Dr. Morris reported that hydroxylation of 
N-2-fluorenylacetamide (2-FAA) in positions 1, 
3, 5, or 7 reduces its carcinogenic potency, and 
the in vivo hydroxylation that occurs is inter- 
preted by Dr. Morris as a detoxification mecha- 
nism or a means of solubilization to facilitate ex- 
cretion (these compounds occur in the urine in 
the largest amounts as conjugates of glucuronic 
and sulfuric acids after 2-FAA feeding). Last 
year also, it was reported that replacement of 
the hydrogen atoms on the omega-carbon of the 
acetyl group in 2-FAA by fluorine led to in- 
creased carcinogenic activity. Fluorination of all 
six omega-carbon hydrogens in 2, 7-fluorenylene- 
Jw-acetamide (2, 7-FAA) has now been accom- 
plished, and this new compound shows increased 
carcinogenicity. Sites in the rat most affected 
were the mammary and salivary glands. It has 
also been shown that the weak carcinogen, N-l- 
fluorenylacetamide (1-FAA), was converted into 
a moderately active one by substitution of the 
hydrogens of the omega-carbon by fluorine; ac- 
tivity was again most pronounced in the mam- 
mary and salivary glands. 

Four papers (with Drs. H. L. Stewart and K. 
Snell) setting forth a lar^e series of observa- 
tions on the carcinogenic effects in rats of 2, 7- 
FAA, including comparisons with 2-FAA, have 
been accepted for publication. Tumors were pro- 
duced in the small intestine, glandular portion of 
the stomach and salivary glands, and neurogenic 
tumors were noted. 2, 7-FAA also produces 
atrophy of many organs and tissues and raises 
questions anew regarding the role of atrophy in 
tumorigenesis. Of special interest is the gastric 
adenocarcinoma produced in the rat, for this is 
the first instance that this important type of can- 
cer has been produced by an orally administered 
carcinogen. 

Animal experiments concerned with the effects 
on carcinogenicity brought about by blocking the 
most active hydroxylation sites in a simp^ aro- 
matic amine such as aniline or acetanilide have 
been partially completed. Blocking both the or- 
tho positions and the para position with -CH 3 , 
—CI and — F was studied as well as the para 
position with -OH. The 2, 4. 6-trifluoroacetanil- 
ide experiments are still incomplete. Liver tu- 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mors were induced in rats fed 2, 4, 6-trimethyl- 
aniline, and a number of animals showed severe 
cholangiofibrosis. Enlarged pituitary glands 
were also noted. In the p-hydroxyacetanilide 
experiment, mammary glands were found to be 
hypertrophied, and a large number of animals de- 
veloped enlarged pituitary glands. Enlarged 
pituitary glands were also seen in rats fed 2, 4, 6- 
trichoroacetanilide and in female rats fed ace- 
tanilide. 

One of the hepatomas (Morris hepatoma 5123) 
recently developed by Dr. Morris is of consider- 
able interest since its enzymatic characteristics 
are very similar to those of normal liver and are 
in fact more similar to normal liver than any 
other transplantable rat tumor studied to date. 
Some enzymatic activities are actually greater in 
the tumor than in normal liver. Dr. Morris is 
collaborating with investigators in several lab- 
oratories in studies on this tumor, particularly in 
regard to Dr. Van Potter's deletion hypothesis. 
This hepatoma was produced in the Buffalo strain 
of rat by a slow acting carcinogen, N-(2-florenyl) 
phthalamic acid (2-FPA). 

Dr. H. M. Dyer, working with Dr. Morris and 
Miss B. Ensfield, has continued her studies on the 
interference of 2-FAA and related compounds 
with tryptophan metabolism. It appears that 
the excretion of increased amounts of urinary 
xanthurenic acid, kynurenic acid, and kynurenine 
derivatives after a load dose of L-tryptophan to 
rats maintained on carcinogenic concentrations of 
2-FAA is due to an inhibition of kynureninase 
activity. Inhibition may result from combina- 
tion of the coenzyme pyridoxal phosphate with 
fluorenamine or some other amino metabolite of 
2-FAA in the form of a Schiff base. Therefore, 
a more than ordinary requirement of vitamin B 6 
may be needed by rats ingesting aminocarcino- 
gens. A relatively low concentration of dietary 
B 6 was found to cause an increase in the induc- 
tion period and/or a low incidence of tumors of 
the liver induced by 2-FAA. Low dietary levels 
of vitamin B 6 inhibited mammary tumor devel- 
opment. 

Drs. J. H. and E. K. Weisburger, with Drs. 
Morris and K. Suzuki and Mr P. H. Grantham, 
continue to make many contributions to our 
knowledge of the mechanisms of carcinogenesis 
and the chemistry and metabolism of N-2-fluor- 
enylacetamide and related compounds. Previous 



work had indicated that monohydroxylation is 
an important biochemical reaction undergone in 
the body by compounds such as N-2- and N-3- 
fluorenylacetamide. The suspicion that the more 
polar metabolites excreted were dihydroxy deriva- 
tives has been confirmed through studies per- 
formed with the synthesized 2, 7-dihydroxyde- 
rivatives of N-3-fluorenylacetamide. In addition, 
N-(7-hydroxy-3-fluorenyl)acetamide formed a 
sizeable portion of the urinary metabolites and 
was the only product excreted as a sulfuric acid 
conjugate. The 2, 7-dihydroxyderivative was ex- 
creted mainly as a glucuronide, as was also the 
previously known 2-hydroxy derivative. The 
polyhydroxylated compounds posses a number of 
charged reactive groups which make them espe- 
cially interesting from the biochemical and bio- 
logical standpoint. 

Studies were continued on the protein binding 
of 2-FAA and its metabolites. It is obvious that 
this kind of binding represents a complex series 
of reactions involving a number of tissue con- 
stituents and several metabolites of the carcino- 
gen. Last year, reactions were described that in- 
volved a few selected metabolites of 2— FAA, 
namely, the aminofluorenols, which gave rise to 
rat liver protein-bound radioactivity as an arti- 
fact. Through use of in vitro and in vivo ex- 
periments involving proteolytic enzymes, it has 
been shown that the aminofluorenols form only a 
small part of the metabolites of 2-FAA and that 
the artifact type of binding plays only a minor 
role in the binding noted in in vivo experiments. 
Studies are continuing on this complex problem. 

Protein binding of 2-FAA was studied in rats 
bearing the Morris hepatoma 5123 with radioac- 
tivity labeled carcinogen. Since this tumor re- 
sembles normal liver in many respects, it was of 
interest to find that proteins derived from this 
tumor showed only about 10 percent of the radio- 
activity of proteins derived from normal livers. 

Drs. E. K. and J. H. Weisburger have per- 
formed considerable synthetic organic chemical 
work to prepare compounds suspected of being 
intermediates in the metabolic sequence, labeled 
carcinogens and their metabolites for distribution 
studies, and carcinogens and derivatives for feed- 
ing experiments. Not only is knowledge in- 
creased in a segment of organic chemistry 
through such studies, but several new reactions 
have been developed during the course of these 



NATIONAL CANCER INSTITUTE 



25 



syntheses and many valuable compounds prepared 
have been made available to several investigators, 
both within NIH and elsewhere. In addition, 
physical-chemical data have been acquired by 
means of ultraviolet and infrared spectra and by 
measuring ionization constants of aromatic 
amines. Polynuclear aromatic amines have been 
synthesized and some show metal chelation. 

Last year Dr. H. F. Blum reported on a model 
that is consistent in a quantitative sense with the 
existing data on cancer induction by ultraviolet 
light (a book by Dr. Blum on this subject has 
been well received by cancer investigators and 
other scientists). Dr. Blum has now formulated 
a quantitative model for carcinogenesis by chem- 
ical agents. This model, although containing the 
same fundamental assumptions as the former one, 
entails equations of a different form, explainable 
on the basis of temporal relationships of the car- 
cinogenic agent. Preliminary examination of the 
goodness of fit of the model for data in the litera- 
ture indicates good approximation. The model 
also points up difficulties in testing the "specific" 
effectiveness of a given carcinogen, since multiple 
factors, which need evaluation in testing, are in- 
volved, some of which are difficult or impossible 
to measure by the usual testing methods. In par- 
ticular, the setting of tolerance levels based on the 
usual type of data obtained in toxicological test- 
ing would likely lead to a false sense of security. 
The new model, like the older one, describes a 
cumulative, essentially irreversible and essentially 
nonthreshold process. 

Viruses 

The Virus Oncology Section, Laboratory of 
Biology, has an integrated program of research 
on viruses and cancer which in toto has a wide 
variety of experimental approaches aimed at elu- 
cidating the role of viruses in human cancer and 
which, at the same time, permits the individual 
senior investigators to work with model animal 
systems of particular interest to each of them. 

"Vtrological Aspects op Human Tumors. From 
knowledge of the tumor viruses of laboratory 
animals, it appears that human tumor viruses, if 
they exist, are likely to be species specific under 
most circumstances. Six major approaches, de- 
signed to take into account this difficulty, are un- 
der way in the attempt to obtain data relevant 



to the question of the viral etiology of human 
cancer: (1) Attempts to detect cytopathogenic or 
other changes in tissue culture following inocula- 
tion of cell-containing and cell-free preparations 
of human cancer tissues into cultures of cells 
from human beings and animals; (2) Attempts 
to detect changes in the chorioallantoic mem- 
branes and allantoic cavities of eggs inoculated 
with cell-containing and cell-free preparations of 
human cancer tissues, and study of the phe- 
nomenon of viral interference in embryonated 
eggs utilizing known chicken or human viruses 
that grow readily in such eggs; (3) Primary in- 
oculation into newborn mice of microsome frac- 
tions of human cancer tissues prepared by the 
methods found most suitable for the Rous sar- 
coma virus; (4) Tests in newborn mice on nu- 
cleic acid fractions derived from human cancer 
tissues; (5) Procedures for producing immuno- 
logical tolerance in tests for human viruses in- 
volving the inoculation of other animal species; 
and (6) Electron microscope studies (in collab- 
oration with Dr. A. J. Dalton). 

The tissue culture studies, which have been in 
progress for about two years, have yielded no 
evidence thus far of a virus of any type being 
associated with human cancer tissue. A total of 
75 separate cancers have been investigated in tis- 
sue culture. With the other approaches, insuffi- 
cient time has elapsed to expect definitive in- 
formation. It is of interest to point out that 10 
cancer specimens have been employed in coor- 
dinated experiments involving all or most of the 
various approaches listed above. 

Because it appears to be of great importance 
in studies on the possible viral etiology of human 
tumors to be able to manipulate artificially a 
virus or host to permit the growth of a foreign 
virus, Dr. M. A. Fink has been attempting to in- 
duce in mice a recognizable, reproducible change 
by the injection of a known, alien tumor virus 
(the avian Rous sarcoma virus) in the hope that 
a useful model system might be developed. Al- 
though hundreds of mice have received injec- 
tions by a variety of routes, no effects have been 
noted. When attempts to prevent an antibody 
response in later life were made by injecting the 
neonatal mouse with a chicken tissue component, 
with cortisone, or with both, it was found that 
cortisone, but not the neonatal injection of 
chicken protein, could completely suppress the 



26 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



formation of antibody to chicken protein. Pre- 
liminary results with administration of Eous sar- 
coma virus to cortisone-treated mice suggest that 
the virus is affecting the animals adversely, but 
more careful control of cortisone dosage is needed 
in further work. 

Extracts of 45 human tumors have been inocu- 
lated into cultures of human cells and into new- 
born and fetal hamsters by Dr. A. Rabson, and, 
thus far, no tumors attributable to the inocula 
have been observed. In one case a cytopathic 
effect was seen in culture, and the agent produc- 
ing the effect was adapted to grow in HeLa cell 
cultures where cytopathic effects were also noted. 
Attempts at identification are under way. 

Dr. S. Stewart is also studying cell-free prepa- 
rations from human neoplasms. Hamsters were 
injected when newborn either with fluids from 
embryonic tissue cultures which had received neo- 
plastic material or with fluids from noninocu- 
lated control cultures. A wide variety of tumors 
resulted in a high percentage of animals (many 
animals had more than one tumor) not only in 
the experimental group, but surprisingly also in 
the control group (essentially the same tumor 
types with the same incidence). Liver cysts, 
spleen atrophy, enteritis and polyps of the bowel 
were also seen in many of the animals. Seven 
percent of the females developed a trophoblast- 
like lesion of the uterus. Results obtained in 
mice which were inoculated for another purpose 
were similar. Of 276 Swiss mice over 12 months 
of age that had been inoculated when newborn 
with culture fluids from control mouse embryo 
cells, 56 percent developed several types of tu- 
mors. Of 103 litter mates that received culture 
fluids plus rabbit antisera (containing antibodies 
to mouse embryo cells), however, essentially no 
unusual tumors were seen. Dr. Stewart postu- 
lates that the high incidence of neoplasms re- 
sulted from an induced tolerance to embryonic 
cells brought about by the tissue culture inocu- 
lum. 

Moloney Virus. Dr. J. B. Moloney continues to 
add to the knowledge of the murine lymphoid 
leukemia virus which he isolated from a trans- 
plantable Sarcoma 37 that had been transferred 
to BALB/c mice after 195 tumor generations in 
strain A/Ln mice. He has now isolated the 
virus from two other transplant lines of Sarcoma 



37, the original tumor line in A/Ln mice and the 
sarcoma line from which this arose, now being 
carried in ZBC mice. Further improvements in 
concentration and purification of the virus have 
been made by means of protamine precipitation, 
followed by trypsin digestion of the protamine- 
virus complex. Regardless of the route of inocu- 
lation, the neoplasm produced is always of the 
generalized type with no tumors at the site of 
inoculation. The intravenous route produces leu- 
kemia deaths in weanling and adult mice about 
one month earlier than those produced by other 
routes. The virus is capable of eliciting a 100 
percent leukemia response in BALB/c mice in- 
oculated as late as 8 months of age. The potency 
of the virus has increased with successive pas- 
sages through newborn mice; after 12 passages 
the average latent period for 100 percent induc- 
tion of lymphocytic leukemia has fallen from 6.4 
months to 2.5 months period. A high percentage 
of animals develop the disease as early as 5.5 
weeks after inoculation. 

Recent studies by Dr. Moloney indicate the 
following important results: the viral induction 
of lymphoid leukemia in the rat; a lymphoid 
leukemia virus that can cross the species barrier ; 
a virus that is not rat strain specific. The agent 
has not acquired host specificity after repeated 
virus passage in the rat or mouse, since virus 
from either rat or mouse passages will induce the 
disease in both species of animals. 

Either thymectomy or splenectomy delays the 
onset of leukemia in the inoculated hosts al- 
though all eventually die with the disease. Thy- 
mectomy of the host followed by inoculation re- 
sults in the induction, in a high percentage of 
cases, of a HodghirCs diseaselike lesion which is 
a reticulum cell neoplasm of Type B as described 
by Dr. T. Dunn. Splenectomy of the host fol- 
lowed by inoculation results, in a few cases, in 
the induction of myeloid leukemia which can be 
further differentiated as chloro-leukemia, an ex- 
tremely rare tumor for this strain of mouse. Cell- 
free material was prepared from the induced 
myeloid leukemia and inoculated into intact new- 
born BALB/c mice; all the mice died with lym- 
phoids leukemia. 

Preliminary results grive good evidence that the 
a^ent is composed of RNA-containing particles. 
Dr. Moloney has initiated experiments to learn 
if the nucleic acid isolated by the method of 



NATIONAL CANCER INSTITUTE 



27 



Gierer and Schramm from a leukemic tissue 
microsome fraction containing active virus can 
induce tumors. 

Preliminary studies by Dr. Moloney indicate 
that the tumor-producing activity of the leu- 
kemic ascites cells, which contain active virus, 
was completely inhibited by oxidation products 
of linolenic acid. 

Dr. T. Dunn, working with Dr. Moloney, has 
found that leukemia first appears in the thymus 
gland in mice inoculated with the Moloney virus. 
Prior to the development of this lesion there was 
noted regularly a marked hyperplasia of the 
spleen and occasionally a localized reticulum cell 
hyperplasia of the lymph nodes. Transplants of 
the hyperplastic spleens from mice with no evi- 
dence of a true leukemia reproduced the same 
sequence of lesions observed after inoculation of 
the virus material, indicating the presence and 
multiplication of the leukemia virus in a host 
which showed no overt signs of lymphoid leu- 
kemia. 

It has been observed earlier that the young 
mice born to leukemic parents developed "spon- 
taneous" lymphoid leukemias at a high incidence 
level after a rather extended period of time. 
Drs. Moloney and N. Ida (M. D. Anderson Hos- 
pital, Houston) have undertaken studies to de- 
termine the mode of transmission of the Moloney 
virus to the offspring. Pregnant mice were in- 
oculated intravenously at various stages of gesta- 
tion. When the virus was given early in gestation 
(1 to 4 days) , a high rate of abortions or resorp- 
tion of fetuses occurred. A high incidence of leu- 
kemia was noted among the young born of the 
group of mice that had received the virus after 
the placenta was formed, with average latent 
periods of 31 to 52 weeks. 

Dr. A. J. Dalton, with Drs. Moloney and W. W. 
Oppelt, has found by means of electron micro- 
scopy that in an analysis of Moloney leukemia 
the primary site of formation and apparently the 
majpr sou rce of virus are the megakaryocytes of 
the bone marrow and spleen. The majority of 
particles in any one megakaryocyte were in the 
same stage of development, the stages varying 
from one cell to the next. Platelets from rats 
rendered leukemic with the Moloney agent were 
examined, Many platelets were found which con- 
tained particles, occasionally centrally located. 



Megakaryocytes and platelets of control rats were 
found to be negative for the particles. 

Dr. H. Kahler and Mr. B. J. Lloyd, in collabo- 
ration with Dr. Moloney, utilizing density gra- 
dient centrifugation and electron microscopy 
techniques, observed that the highest concentra- 
tion of Moloney virus particles of the 100 milli- 
micron diameter size was found at the level in 
the centrifuge tube where the density was calcu- 
lated to be 1.24. 

Gain in knowledge of the murine tumor caus- 
ing viruses would be greatly facilitated if more 
rapid methods of assay not requiring long latent 
periods of direct observation in animals were 
available. Dr. F. J. Rauscher is seeking to im- 
prove assay methods by attempts to increase the 
potency of the viruses under study, reduce or 
remove inhibitors present in some assay systems, 
and develop simplified assay, systems, e.g., in 
embryonated chicken and Japanese quail eggs. 
One approach showing success is an assay of the 
Schoolman-Schwartz mouse leukemia virus in- 
volving the virus interference phenomenon. The 
leukemia virus has been serially passaged on the 
chorio-allantoic membrane (CAM) of embryo- 
nated hens eggs. No discrete lesions were ob- 
served grossly or histologically; however, when 
eggs were inoculated with CAM extracts of the 
first and sixth serial passages and then chal- 
lenged with influenza virus, a 90 to 99 percent 
reduction in the titer of the challenge virus was 
noted. The same CAM extracts when heated to 
60° C. for 30 minutes, or extracts of CAM's from 
eggs inoculated with normal mouse tissue failed 
to depress the titer of influenza virus. Extracts 
of CAM's showing this interfering activity have 
not, however, produced leukemia in mice, as of, 
an approximately 2-months' postinoculation pe- 
riod. 

Similar studies are under way with the Moloney 
virus. It is hoped that these methods can also 
be adapted to studies with human material as 
further successes are achieved. 

Dr. R. A. Manaker has made additional ad- 
vancement in the in vitro propagation of the 
Moloney virus. With improved culture media 
and primary mouse spleen cultures, he has found 
that the fluids overlying the cultures are infective 
for both animals and fresh spleen cultures, pro-, 
vided the cultures are maintained at least two 
weeks following addition of cell-free extracts of 



28 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



spleen, thymus and lymph node pools obtained 
from leukemic mice. The virus does not prolif- 
erate as rapidly when inoculated into long-term, 
cultures, but will do so sufficiently to produce 
leukemia in test animals if maintained for as 
long as three months. Tests for infectivity must 
be evaluated in terms of the tumor-inducing 
ability in animals since no cytopathogenic effects 
are seen in the cultures, even if maintained with- 
out transfer for as long as three months. Tissue 
culture fluids containing the virus may be stored 
at —70° C and used to infect fresh cultures at a 
later date. Leukemia has not been observed in 
mice inoculated with control spleen culture fluids. 
On the other hand, leukemia has been observed 
in uninoculated mothers and uninoculated litter 
mates of mice infected with active virus material, 
suggesting that the virus may be contagious. Dr. 
Manaker's successful propagation of the Moloney 
virus demonstrates that its in vitro behavior is 
similar to that of other animal viruses, and the 
findings add support to the argument that aside 
from the type of response they elicit in the host, 
the tumor viruses are not basically different from 
other viruses. 

Drs. Fink and Moloney are investigating the 
immunological properties of the Moloney virus 
and have produced the following homologous and 
heterologous sera : normal mouse and normal rab- 
bit; mouse antinormal spleen; rabbit antinormal 
spleen; mouse antivirus (formalin-killed); and 
rabbit antivirus (purified). Only the mouse 
formalin-killed virus antiserum proved to be 
effective in neutralizing the leukemogenic activity 
of the virus. With this antiserum, an indication 
has been obtained that complement fixation does 
occur in low titer. 

Polyoma Virus. Dr. L. W. Law, alone and with 
collaborators, has been particularly active in 
studying biological parameters of polyoma vi- 
ruses. With Dr. H. Kahler and Mr. B. Lloyd, 
Dr. C. Dawe and Drs. W. P. Rowe, and J. Hart- 
ley (LID, NIAID), Dr. Law has found four 
strains of virus to be highly variable in a variety 
of biological properties, including tumor induc- 
tion, a result not unlike findings obtained with 
other viruses such as the Rous sarcoma virus and 
the mouse mammary tumor agent. A highly 
virulent virus strain required only 20 to 30 par- 
ticles for tumor formation; on the other hand, 



another required more than 1 million particles. 
Different dose levels of the highly virulent strain 
give different patterns of tumor induction. 
Dawe's strain, adapted for growth on P-388 cells 
in high-serum medium, undiluted and in 10 2 dilu- 
tion produces thymic epithelial tumors while in 
10 4 and 10 6 dilutions produces unilateral parotid 
tumors. The weakly oncogenic strain of Rowe 
(397), on the other hand, produces parotid tu- 
mors only in the undiluted extract. 

C57BL/ Ka strain mice show a pronounced re- 
sistance to tumor induction by a virulent strain 
of polyoma virus. After an observation period 
of 6 months, 92 percent of C3Hf/ B i mice have 
developed thymic epithelial (and other) tumors 
while none of the C57BL/ Ka mice has developed 
tumors. Only 10 percent of Fi mice have been 
tumorous. 

Drs. Law, Dawe and Rabson have found differ- 
ences in infectivity and oncogenic potentialities 
between polyoma grown on P-388 cells cultured 
in milk medium and polyoma grown on P-388 
cells grown in human serum medium. The for- 
mer retained high infectivity for mouse and for 
tissue culture but was nearly nononcogenic, 
whereas the latter, with a reduced infectivity, 
remained highly oncogenic. Nevertheless, the 
virus grown on the milk-adapted cells retained 
its oncogenicity for hamsters. 

Drs. Law and Dawe have observed the appear- 
ance of several neoplasms in mice into which 
polyoma infected fragments of cultured salivary 
gland tissues (30-50 days on a sponge-matrix 
system) had been placed, both in X-rayed adults 
or newborn animals. In extensive studies of 
three of the tumors, no virus could be detected 
in two fibrosarcomas, even through eight trans- 
fers of tissue in mice, but it was found to be 
replicating in an epithelial tumor. The results 
are similar to those reported by Dr. Dulbecco 
(Cal. Tech.) and by Dr. Sanford (NCI) in their 
studies of fibrosarcoma development in vitro. 

Dr. Law, also with Dr. Dawe, has investigated 
the effects of X-irradiation, administered prior 
to introduction of polyoma virus into adult 
C3Hf/ Bi mice. Thirty-eight and 43 percent of 
the adults presented single or bilateral parotid 
gland tumors following intravenous introduction 
of thymotropic polyoma virus, a result strikingly 
different from that of the same strains infected 
in the neonatal period. Thymectomy of adults 



NATIONAL CANCER INSTITUTE 



29 



had no influence on the carcinogenic response and 
virus or X-rays alone were ineffective. X-irradi- 
ation plus natural infection (at 3 to 5 months of 
age) did not induce neoplasms characteristic of 
this virus. Thymic tumors induced by the virus 
seldom grew when transplanted into suitable 
hosts, either in young mice or in X-rayed adults. 
One of the tumors that did develop was strikingly 
hormone dependent. 

Dr. C. Dawe has obtained additional informa- 
tion as part of the overall study on influences of 
host factors on the development of cytolytic and 
proliferative responses to polyoma virus in vitro. 
With Dr. Law, he has found that cultures of 
salivary gland tissue from adult mice showed an 
epithelial proliferative response greater than that 
seen in cultures of this tissue from newborn mice. 
At the 30-day interval after infection, cultured 
salivary gland rudiments from 13- and 14-day 
old mouse embryos, however, showed less epithelial 
proliferative response than that seen in cultures 
from either newborns or adults. No proliferative 
response by mesenchymal cells was seen, and 
necrosis of these cells was observed in glands of 
all ages. As indicated above, nearly half the 
adult mice receiving polyoma virus following 
total body radiation develop salivary gland tu- 
mors, even though no such tumors developed in 
animals treated with X-ray alone nor in adult 
nonirradiated controls. This information, cou- 
pled with early results on the effects of specific 
antibodies on cultures obtained in collaboration 
with Dr. W. Kowe (NIAID), plus the knowl- 
edge that antibody response is delayed in newborn 
mice, indicates that the high susceptibility of 
newborn mice to polyoma virus tumor induction 
depends primarily on the weakness of the immune 
response at this age and not on the presence of 
"primitive" cells or on a higher susceptibility of 
rapidly growing cells. 

Dr. Dawe has shown that the response of 
salivary glands from rats, hamsters, mastomys 
and human fetuses grown in culture following 
exposure of the excised tissue to polyoma virus 
was negative, the changes previously reported in 
similar mouse salivary gland cultures not being 
observed. 

Dr. S. E. Stewart, in collaborative studies with 
Drs. C. and R. Leuchtenberger (Children's Hos- 
pital, Boston), has confirmed and extended pre- 
vious observations with polyoma virus indicating 



two main processes in tissue cells of the kidney : 
(a) a degenerative process in the epithelial cells 
associated with the viral activity leading to de- 
struction of cells, and (b) a proliferative process 
in the stroma without evidence of viral activity, 
but associated with cellular activity leading to 
further propagation of abnormal cells resulting 
in tumors. The epithelial cell changes preceded 
the proliferation of the stromal cells. It was 
concluded that only the stromal cells become ma- 
lignant and, on the basis of histochemical deter- 
minations of DNA, that viral proliferation oc- 
curred only in the epithelial cells. This result 
appears to be in accord with Dr. Stewart's pre- 
vious observations since no renal carcinomas have 
been found in mice injected with polyoma virus, 
but renal sarcomas are common. Dr. Stewart 
suggests that a cell responds to the virus in one 
of two ways: (a) in one instance the cell is used 
by the virus for its replication, suppressing cell 
mitosis and finally killing the cell; and (b) in 
the other instance the virus or its products acts 
as a stimulating agent provoking reduplication 
of cells resulting in malignancy. 

Drs. E. Love and A. Rabson and Miss F. 
Legallais have determined a sequence of changes 
in polyoma-infected P388Di cells grown in cul- 
ture by means of cytochemical techniques. The 
earliest abnormalities seen consist of enlargement 
of the nucleus, nucleolus and nucleolinus and the 
appearance of intranuclear vacuoles in which nor- 
mal chromatin and ribonucleoprotein are replaced 
by abnormal histone-free deoxyribonucleoprotein. 
Other changes in nucleoprotein become evident 
in the nucleus, and terminally, alteration in the 
cytoplasm is observed. 

Drs. W. Banfield and C. Dawe have found that 
virus-like particles can be seen easily in polyoma- 
induced mouse tumors kept in a maintenance 
media in tissue culture for as little as 24 hours, 
but cannot be found after a reasonable amount of 
searching in tumors examined directly. Several 
human tumors held in tissue culture from 24 
hours to 2 weeks produced negative results. With 
Dr. W. Rowe (NIAID), initial electron micro- 
graphs were made of mouse adenovirus (morpho- 
logically in the adenovirus group) and mouse 
thymic agent (features in common with the 
Lucke, salivary gland, herpes simplex and herpes 
B viruses). 

Dr. M. Stanton has studied the histopathologic 



30 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



characteristics of the polyoma-induced osteogenic 
tumors in Strain A mice. These tumors, which 
are especially amenable to progression studies 
because of slow, step-wise changes, are of interest 
since Strain A mice receiving polyoma virus de- 
velop them without developing the variety of 
tumors observed in other strains and without 
affecting the incidence of spontaneous pulmonary 
alveologenic tumors. 

Dr. A. Rabson has succeeded in producing tu- 
mors of the lung in hamsters by direct instilla- 
tion of polyoma virus in the trachea. In 6 of 
the 16 animals autopsied lung tumors with histo- 
logic features of bronchiolar carcinoma, alveolar 
cell carcinoma and squamous cell carcinoma were 
found. The squamous cell carcimona, which 
appeared to arise in bronchioles and intra-pul- 
monary bronchi, has been established as a trans- 
plantable tumor and is now in the 16th trans- 
plant generation. Attempts to isolate polyoma 
virus from this transplantable tumor and from 
one of the primary tumors have been unsuccess- 
ful, an experience similar to that of other investi- 
gators who have encountered difficulties in isolat- 
ing this virus from the sarcomatous lesions of 
hamsters. 

Newborn Mastomys {Rattus natalensis) inocu- 
lated with polyoma virus by Dr. Eabson devel- 
oped tumors in 14 of 17 animals autopsied (sar- 
comata in kidney, heart and subcutaneous tissue 
and angiomatous tumors in liver). Thus far, no 
parotid gland or thymic tumors similar to those 
produced by polyoma virus in mice have been 
observed, however. Polyoma virus was isolated 
in tissue culture from suspension of one of the 
renal sarcomas that developed. 

Mammary Tumor Agent. In a study designed 
to elucidate the factors responsible for the sud- 
den disappearance of the mammary tumor agent 
from a few strain Mil mice, Dr. H. B. Ander- 
vont has obtained further evidence that the virus 
carried by Mil mice is much weaker in tumor- 
inducing capability than that carried by C3H 
mice, thus being one factor of probable signifi- 
cance in the disappearance of the agent. The 
observation that an appreciable incidence of tu- 
mors occurred in F x hybrids derived from agent- 
free (Mil-) females and agent-carrying 
(C3H+) males was of interest. 
Mil virus has been introduced into C3H 



mice and C3H virus into Kill mice by fostering 
nursing, and the animals have been followed now 
for several generations of inbreeding. The C3H 
mice had a low incidence of tumors, and no defi- 
nite signs of increased potency of the weak Mil 
virus were seen. With the Mil mice carrying 
the C3H virus, observations indicate that a single 
strain of virus in hosts of the same genetic con- 
stitution shows a pronounced variation in its 
ability to induce tumors. Even within a highly 
susceptible family, an occasional Mil mouse is 
very resistant even to the potent C3H agent. 

In some cases, tumor-causing viral agents can 
escape through certain sized pores in the mem- 
branes of diffusion chambers placed in the peri- 
toneal cavity of mice. For example, Dr. R. 
Merwin has shown that the mammary tumor 
agent passes through pores 0.45 micra but not 
0.10 micra in diameter, and that the Moloney 
virus passes through 0.45 micra pores, but no 
smaller ones. Even with 0.45 micra pores^ no 
tumors appeared in mammary tumor agent-free 
BALB/c hosts when mammary tissue from agent- 
free C3H donor mice was placed in the chambers, 
a finding providing additional evidence that the 
virus is indeed absent from so-called virus-free 
mice. 

Rous Sarcoma Virus. In attempting to deter- 
mine the ultimate limit to which the concentra- 
tion of Rous sarcoma virus in sarcoma tissue 
can be increased through continued selective 
serial passage in chickens, Dr. W. R. Bryan has 
made such passages, selecting the tumor first to 
appear in each batch of 40 inoculated chickens, 
for the past 5 years at biweekly intervals (ex- 
cept during the past year the interval was 
changed to monthly). By this procedure the 
virus yield of tumors has been increased about 
100-fold, but during the past year little increase 
was observed. It appears unlikely that the yield 
of virus will be increased further by this tech- 
nique, but the virus concentration already 
achieved justifies resumption of studies on puri- 
fication of the virus. 

Drs. Bryan, H. A. Sober, and F. J. Rauscher 
and Mr. J. P. Kvedar are attempting to purify 
further the Rous sarcoma virus obtained from 
"high potency" tumors by means of differential 
ultracentrifugation and chromatography on cel- 
lulose ion-exchange columns. An increase in 



NATIONAL CANCER INSTITUTE 



31 



virus activity, similar to the 100-fold increase 
obtained by the selective passage procedure, is 
present in partially purified fractions separated 
by differential centrifugation or cellulose chro- 
matography. By both procedures there is a 2- to 
3-fold increase in viral activity per gram equi- 
valent of tumor tissue, indicating that an "in- 
hibiting" factor is removed. 

Drs. Rauscher and M. A. Fink have studied 
the effects on the production of infective Rous 
sarcoma virus (RSV) by conditioning the host 
with Freund's adjuvant. When the adjuvant was 
given to young chicks before virus inoculation, 
extensive granulomatous lesions developed at the 
site of injection (subcutaneous) ; when a small 
dose of RSV was injected into these lesions, the 
granulomas grew at an accelerated rate and ap- 
peared to invade adjacent muscle. Extracts of 
these growths yielded 2 to 4 logs of virus. 
The same small dose of virus failed to produce 
tumors in control birds pretreated with saline, 
and RSV could not be demonstrated in tissue ex- 
tracts from these control animals. The incidence 
and severity of metastases were also increased in 
birds pretreated with adjuvant. When chicks 
were inoculated with a dilution of RSV that 
failed to produce tumors and were subsequently 
inoculated with adjuvant, 85 percent developed 
relatively large amounts of anti-RSV antibody in 
their sera. The sera of control negative birds 
challenged with saline remained free of signifi- 
cant levels of antibodies. 

Although Dr. Rauscher has failed to effect a 
recovery of infective RSV from RSV-specific 
antibody complexes by treatment with fluorocar- 
bon (Genetron 113) , he has done so by tech- 
niques of ultracentrifugation and under certain 
conditions with simple dilution. Through spe- 
cial techniques involving trypsin he has also 
made noninfective lesions produced by low doses 
of RSV or noninfective lesions in Japanese quail 
eggs yield infective RSV. 

These techniques that are opening leads for en- 
hancing host response to a virus, for preparing 
more potent virus, for detecting and eliminating 
inhibitors, and for measuring responses produced 
by minimal amounts of virus are particularly 
important for the study of the initiation and 
course of viral diseases as they exist in nature 
and are especially germane in laying the ground- 



work for investigation of the viral etiology of 
human tumors. 

Other Virus Studies. In collaboration with 
Dr. J. A. Reyniers (Germ-Free Life Research 
Center, Tampa, Florida) and Mr. J. P. Kvedar, 
Dr. Rauscher is evaluating the value of Japanese 
quail (Cotumix coturnix japonica) embryonated 
eggs and posthatched chicks in virus and cancer 
research. Their small size before and after 
hatching, their ease of maintenance, and their 
rapid generation time makes them especially 
suitable as an avian host in studies requiring 
long periods of observation, particularly since Dr. 
Rauscher has shown that they may be manipu- 
lated in a manner similar to chickens and that 
they are susceptible to at least six different 
viruses. In addition, certain interesting differ- 
ences have been noted with the Rous sarcoma 
virus and with the visceral lymphomatosis virus. 
Of special interest is the finding of a substance 
inhibitory to the Rous sarcoma virus growth in 
eggs that failed to develop gross lesions. The 
possible relationship of this phenomenon to "in- 
terferon," to the resistance-inducing-f actor of 
Rubin, and to lymphomatosis virus is under 
study. 

More knowledge is needed on the indigenous 
mouse viruses because, among other reasons, they 
may interfere with the expression of tumor-caus- 
ing viruses under study, they may produce disease 
in colonies of study animals thus invalidating 
experiments in progress, and they may, if suffi- 
cient information can be gained, serve as models 
of tumor virus behavior involving latent infec- 
tions. Dr. R. A. Manaker has discovered three 
such viruses (two that produce hepatitis and one 
that produces pneumonia) and has determined 
several of their characteristics. Such informa- 
tion is useful in the control of the diseases pro- 
duced in colonies by these viruses and provides 
basic knowledge needed for study of the tumor 
viruses in animals that are potential carriers of 
these agents. 

Drs. C. Dawe and L. Kilham (DBS) did not 
find a proliferation-promoting effect of rat virus 
in organ cultures of rat salivary glands, a system 
analogous to the polyoma virus-infected mouse 
salivary ffland culture system. 

Drs. W. Banfield and G. Ramirez and Mrs. D. 
Brindley, in studies with a spontaneous reticulum 



32 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cell sarcoma in a hamster now carried through 
12 passages, have shown that while injections of 
blood, ascites fluid, and brain from tumor-bear- 
ing animals produce tumors in the recipient ham- 
ster, cell-free filtrates of brain, ascites fluid, and 
tumor so far have not. Of six animals fed fresh 
tumor tissue, one developed systemic tumor in- 
volvement and another laryngeal occlusion from 
submucosal tumor growth. More remarkably, of 
10 untreated animals caged with 10 animals in- 
oculated with tumor, six died with laryngeal ob- 
struction caused by submucosal tumor growth 
and two killed animals had systemic tumor in- 
volvement. The tumor also arose in one of three 
hamsters separated by a screen from tumor-bear- 
ing animals. As yet, no viruslike particles have 
been seen upon examination by electron micro- 
scopy. 

In electron micrographs, Dr. R. F. Zeigel has 
observed type A "virus particles" of Bernhard 
budding from the apical plasma membrane of 
pancreatic acinar cells in the chick. Although 
the viral nature of these particles has not been 
established, they possess morphological charac- 
teristics closely similar to those described in asso- 
ciation with lymphomatosis, Rous sarcoma, my- 
eloblastosis and other avian viral infections. 
When particles were observed to bud from pan- 
creatic acinar cells, all other tissues examined 
contained the particles in intercellular spaces and 
in intracytoplasmic vesicles and vacuoles. When 
absent in the pancreas, they were not seen in 
other tissues. In a study of the albumin-secret- 
ing gland of the hen oviduct, the secretory prod- 
uct appears to be produced in close association 
with the ergatoplasm, accumulated in large cis- 
ternal regions and released into the secretory 
lumina. 

Mr. B. G. Young and Dr. P. Mora are con- 
ducting fundamental studies on bacteriophage 
and are preparing to apply some of the tech- 
niques developed to the tumor-causing virus, 
polyoma. Such studies are part of a program 
designed to study interactions of biological sys- 
tem at the molecular level and employ charac- 
terized, synthetic positive (basic) or negative 
(acid) derivatives of polysaccharides with differ- 
ent degrees of branching and charge distribution 
(vide infra) . The bacteriophages are being used 
as model systems to study quantitatively the 
changes that polyelectrolytes may bring about in 



the surface proteins of the virus, in order that 
more can be learned of effects on tumor-causing 
viruses, particularly in regard to blockage of the 
infective process. 

The extent of inactivation of viability of bac- 
teriophage (by blockage of attachment of the 
phage to the bacterial cell and/or the injection 
of DNA into the bacterial cell) was determined 
by means of incubation of the virus with nega- 
tively charged synthetic polysaccharide deriva- 
tives. Inactivation of bacteriophage by polyglu- 
cose sulfate, which occurs at an acidic pH range 
in which sulfuric acid does not produce inactiva- 
tion, was not reversed by further incubation at 
neutral pH, by increasing the salt concentration, 
or by adding positively charged macromolecules. 
Thus, the inhibition is not due to a purely electro- 
static interaction with the positive lysozyme-like 
protein in the tail of the phage. That an ion 
exchange effect was not producing the inactiva- 
tion was shown by inactivation even in the pres- 
ence of high calcium ion concentrations. Fur- 
thermore, since polyglucose sulfate, labeled with 
S 35 , interacted with the T2 bacteriophage directly 
and did not penetrate into the E. coli cell, the 
mechanism of inactivation is most likely due to 
prevention of normal attachment or DNA in- 
jection, rather than blocking processes of viral 
multiplication in the cell. 

When the inactivation was only partial, the 
residual viable phage had a normal attachment 
rate to the E. coli. The attachment of the total 
phage components also was not reduced signifi- 
cantly. These conclusions were based on measure- 
ments done with P 32 -labeled DNA and S 35 -labeled 
protein of the phage. Experiments of shearing 
off the labeled attached phage from the E. coli 
surface by swirling it in a Waring blendor 
showed that it was easier to shear off DNA from 
the inactivated phage than from an untreated 
virus. No incorporation of labeled P 32 into the 
progenies of the bacterial phages was found. 

A reasonable interpretation of the above re- 
sults is that the lysozyme-like protein is blocked 
by the anionic polysaccharide, but not sufficiently 
to prevent attachment, only the penetration and 
injection of the DNA. It is possible that a sec- 
ond inactivation mechanism might also occur, 
possibly simultaneous with the other mechanism. 
Negative DNA in the head of the virus is known 
to be partially neutralized by positive oligopep- 



NATIONAL CANCER INSTITUTE 



33 



tides such as putrescine (findings of Dr. B. Ames, 
NIAMD). The DNA may change its shape if 
the positive putrescine is removed by the nega- 
tive polysaccharide, making eventual passage 
through the narrow tail core impossible. 

It is of interest to note that a small percentage 
of the bacteriophage population which remained 
viable gave progenies that were more resistant 
to further inactivation, suggesting that inactiva- 
tion might be a useful tool for genetic studies. 
Low concentrations of polyglucose sulfuric acid 
were also found to reactivate some of the bac- 
teriophage that had been inactivated with anti- 
body, indicating a direct interaction between the 
antibody and the polyglucose sulfate. Such find- 
ings suggest possibilities of antibody fractiona- 
tion and new ways of studying antigen-antibody 
interactions. 

In the course of preparing for studies on tu- 
mor viruses, Mr. Young and Dr. Mora are modi- 
fying the method of Dulbecco and Sachs, which 
produces plaques in a monolayer of embryonic 
tissue culture, for studies on polyoma. 

Host — Tumor Relationships 

Host Genetics in Carcinogenesis 

Genetics of the mouse has reached a state of 
development where analysis of effects of specific 
genes can now profitably be pursued. There are 
at least 87 gene loci of the mouse identified in 19 
linkage groups, and they may represent all but 
one of the chromosomes. Nine of these loci have 
been shown to be linked with neoplasia. Not 
only has Dr. W. E. Heston over the years made 
major contributions to the knowledge of strain 
differences in relation to cancer, but he has been 
in the forefront in amassing data on the role of 
specific genes of the mouse associated with spe- 
cific types of cancer. He has been particularly 
interested in two loci that are linked with cancer : 
the obese gene ob in linkage group XI and the 
lethal yellow gene A y in linkage group V. The 
ob gene results in excessive obesity, and the A y 
gene is lethal when homozygous, but when hetero- 
zygous causes the coat to be yellow and causes an 
increase in body size. From studies on the latter 
a unique system has been developed which will 
permit new approaches to understanding of the 
mechanism of gene action in mammals, at both 



the molecular and higher organismic levels. Since 
the A y gene is lethal when homozygous, forced 
heterozygosis at this locus during the long period 
of inbreeding in the YBR strain has led to a 
situation in which Fx hybrids resulting from out- 
crosses between mice of the YBR strain and those 
of any other inbred strain are isogenic in that 
they are genetically alike except that half carry 
the A y gene and are yellow and half carry the 
nonagouti allele and are nonyellow. Thus, in 
these Fi hybrids, analysis can be made of the 
effect of this specific gene, for any difference in 
occurrence of tumors in the two color types is 
known to be due to this gene. A further advan- 
tage in these Ft hybrids is that tissues can be 
transplanted freely between the two color groups 
since in Dr. Heston's Laboratory it has been 
shown with skin transplants that the A y gene has 
no histocompatibility effect. This enables in- 
vestigators, after identifying an effect of the gene 
on any type of tumor, to try further to identify 
its path of action through transplantation of tis- 
sues or organs. 

Dr. Heston is employing this system with 
transplantation of ovaries to learn if these or- 
gans are involved in the mechanism of action of 
the A y gene since the gene reduces the age at 
which mammary tumors appear in virgin females 
from 15 to 8 months and since breeding elimi- 
nates the difference between groups of female 
mice with and without the gene. Transplanta- 
tion studies in young adult animals so far have 
failed to implicate the ovaries; transplantations 
in day old Fi hybrids are being done. 

In addition to the increased susceptibility to 
mammary tumors and pulmonary tumors previ- 
ously reported, last year Dr. Heston found that 
males with the A y gene had a significantly higher 
incidence and average number of spontaneous 
hepatomas than those without the gene. The 
feeding of carbon tetrachloride had no detect- 
able carcinogenic effect on inducing hepatomas in 
these highly susceptible male mice. In these Fi 
hybrids the A y gene increased body weight by 
about 10 grams in the males and 15 grams in the 
females. Positive correlations were found in the 
males between the number of hepatomas and body 
weight, body length and length of femur. 

In similar studies on the ob gene, Dr. Heston 
has found that this gene decreases the occurrence 
of spontaneous pulmonary tumors. He previously 



34 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



reported that this gene reduces the incidence of 
induced pulmonary tumors. Male mice with the 
ob gene also show a higher incidence of hepa- 
tomas, and administration of carbon tetrachloride 
gave no differences in occurrence of hepatomas 
between the obese ob mice and the normal sibs. 

Dr. M. K. Deringer previously reported a re- 
duction in the incidence of mammary tumors in 
mice of strain C3He which she produced by trans- 
planting fertilized C3H ova to pregnant uteri of 
C57 black mice without the mammary tumor 
agent and an even lower incidence in DBA/2e 
mice (transfer of fertilized ova from strain 
DBA/2 to C57 BL uteri). Although the data 
are not complete, it appears that forced-breeding 
raises the incidence of mammary tumors in strain 
DBA/2e females, but the incidence does not begin 
to approach the nearly 80 percent figure seen in 
the DBA/2 strain of breeding females. 

In studies of Fi hybrids resulting from out- 
crosses of strain YBK animals (carrying the 
lethal-yellow gene) and strain DBA/2 animals 
(susceptible to the development of leukemia) in 
which the skins were repeatedly painted with 
3-methylcholanthrene solution, Dr. Deringer has, 
as yet, found no effect of the lethal-yellow gene 
on the development of leukemia, but only a small 
number of leukemias have thus far been observed. 
To date, tumors of the skin occurred more fre- 
quently in the yellow than in the brown females 
and about equally in the yellow and brown males 
of the treated groups. 

Radiation Studies 

Dr. L. Law is continuing his investigation of 
the role of variables in X-ray induced and spon- 
taneous leukoses. Chimeras developed in C3H 
mice (both the Law and Bittner sublines) through 
introduction in the neonatal period of AKR bone 
marrow showed the same incidence of X-ray in- 
duced lymphocytic and other neoplasms of reticu- 
lar tissue as the nonchimeric controls, but intro- 
duction of AKR thymic tissue into the chimeras 
increased the frequency of tumors in the Law 
subline, but not in the Bittner subline. Few 
thymic grafts were involved in the neoplastic 
response, and the tumors grew in the host strain 
(C3Hf/L w ) and not in AKR mice. The presence 
of C3H thymus may therefore inhibit the process 
of leukemogenesis. 

Fetal C57 BL mice were resistant to doses of 



X-ray that induced tumors in 24-hour and 1- 
month-old mice. This finding is in agreement 
with that of Upton, who also observed an insen- 
sitivity of fetal tissues in another strain, and 
indicates the desirability of further investigation, 
particularly during those early periods when 
other tissues function as hematopoietic tissues. 

Total body irradiation increases the incidence 
of reticulum cell sarcomas and granulocytic neo- 
plasms in thymectomized C57 BL mice. Shield- 
ing of a thigh, in contrast to this influence on 
thymic lymphoma induction, has been observed 
to be without effect on the induction of these later 
appearing forms of leukoses. Differences in re- 
sponse to fractionated, whole-body X-irradiation 
have been observed among three C3H sublines. 

Miss D. Uphoff has extended her work on the 
genetic factors influencing protection against 
lethal total-body irradiation by a postirradiation 
inoculation of bone marrow to include the mid- 
lethal dosage level. Sufficient data are now avail- 
able so that generalizations and comparisons can 
be made as to the effects of the dose of irradiation 
on the successful transplantation of bone marrow 
at the midlethal and lethal dosage levels. The 
postirradiation inoculation of genetically com- 
patible marrow (marrow donor and host share 
the same histocompatibility -2(H-2) phenotype) 
will result in protection of the irradiated mice 
with no apparent immunological response at 
either dosage level. The available information 
indicates that these protected animals are chime- 
ras consisting of an irradiated host of one geno- 
type and a hematopoietic system of the donor 
genotype. The inoculation of genetically incom- 
patible marrow (marrow donor and host of differ- 
ent H-2 phenotypes or of the H-2 k phenotype), 
gives somewhat different results. Following 
lethal irradiation incompatible marrow may give 
rise to a graft versus host reaction ("secondary 
disease") which may result in death of the irradi- 
ated marrow inoculated mice after apparent re- 
covery from the acute irradiation damage. The 
severity of the reaction and degree of mortality 
varies with the strain combinations and is prob- 
ably a function of the antigenic incompatibility 
of the strains employed. The inoculation of 
genetically incompatible marrow following expo- 
sure to a midlethal dose of irradiation may result 
in what appears to be a host versus graft reaction 
which may result in an early death of the treated 



NATIONAL, CANCEB INSTITUTE 



35 



mice. Although the exact cause of death has not 
been determined, it is clearly a function of the 
phenotype of the irradiated host. It occurs fol- 
lowing the irradiation and marrow inoculation of 
two different strains of the H-2 k phenotype 
(other strains of the H-2 k group have not been 
tested). This lethality of the treatment has not 
been observed in irradiated hosts of the H-2 a , 
H-2 b or H-2 d phenotypes. 

Miss Uphoff has obtained chimeras by exposing 
mice to lethal doses of X-ray and protecting them 
with suitable bone marrow. Under these condi- 
tions the lymphomas arising in the irradiated 
mice were mostly of the donor tissue, and those 
of the host genotype were cases where initial pro- 
tection by donor marrow permitted regeneration 
of the host's marrow. When mice received a 
midlethal dose of irradiation and homologous 
bone marrow, most tumors were of host origin. 
At this dose level lymphomas arise primarily in 
genetic combinations in which the host and donor 
are of different histocompatibility-2 (H-2) phe- 
notypes. In H-2 b or H-2 d combinations in which 
host and donor are of the same H-2 phenotype, 
lymphomas have not been observed. It would 
appear that although the marrow graft is capable 
of protecting the host against the acute irradia- 
tion damage, it does not appear to protect the 
irradiated mouse against this late radiation ef- 
fect, possibly because of the regression of the 
graft as indicated by the lymphomas of host tis- 
sue origin. 

Miss Uphoff had found earlier that amethop- 
terin may protect mice that would normally 
succumb to a homograft reaction following lethal, 
total-body irradiation and homologous marrow 
inoculation. Such mice have a permanent state 
of this type of immunologic nonreactivity since 
it was shown that the mice are chimeras. On the 
basis that treatment with amethopterin in the 
presence of specific antigens might lead to a 
"tolerant" state in the adult mouse, mice were 
treated with the drug and strain specific antigens 
in the form of a crude tissue brei of spleen and 
thymus. Effects were determined by transplanta- 
tion of tumors of the same strain as the antigens. 
Preliminary results are suggestive of success in 
inducing a "tolerant" state since 5 of 15 mice 
receiving both drug and antigen died of pro- 
gressive tumor growth ; mice treated with antigen 
alone were resistant to tumor growth, while with 



drug treatment alone only 1 of 14 had a positive 
test. Because tumor transplantation studies may 
be less adequate for evaluation than other types 
of transplantation procedures and because of the 
urgent clinical need for a method other than 
irradiation for the preparation of the host for 
a homotransplant, other studies are under way 
e.g., on dogs with Drs. Ferrebee and Thomas at 
the Mary Imogene Bassett Hospital. 

Radiation of Cells in Tissue Culture. Dr. M. 
Elkind, in an extension of his previous studies 
dealing with the repair of sublethal X-ray dam- 
age, has found certain experimental conditions 
under which it appears that survival of mam- 
malian tissue culture cells following irradiation 
may be influenced by the proximity of neighbor- 
ing cells. Such a result has important implica- 
tions since one of the basic tenets of radiobiol- 
ogy, the assumption that the site structure in 
cells lethally affected by ionizing radiation is dis- 
crete, requires that a cell in a group of cells 
should survive as an individual and uninfluenced 
by the proximity of its neighbors. 

In an investigation of the influence of drugs 
on lethality and recovery in X-irradiated mam- 
malian cells grown in culture, Drs. Elkind and 
W. Mohler have obtained results with 5-bromo- 
deoxyuridine (BUDR) which indicate: (1) 
BUDR. can increase the X-ray sensitivity of a 
Chinese hamster culture cell line; (2) the increase 
is accompanied by alterations in the survival 
parameters, mean lethal doses (MLD) and extra- 
polation number (a graphically determined esti- 
mate of the loci, or sites, each containing one 
target with the necessary and sufficiency condi- 
tion for survival being the viability of at least 
one locus, in an irradiated cell) ; (3) the effects 
on MLD and extrapolation number may be sep- 
arable under some circumstances; and (4) the 
alteration of sensitivity is probably not merely 
the result of nonspecific cell injury since other 
drugs alter one X-ray survival parameter (MLD) 
without affecting the other. 

Immunological Phenomena and Polysaccharide 
Studies 

Improvements in techniques and increase in 
knowledge of immunological approaches are open- 
ing new avenues of research on host humoral and 
cellular factors involved in natural immunity to 



36 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



infection and neoplastic disease. The polysac- 
charide nature of bacterial endotoxins, their im- 
portance in study of immunologic phenomena, 
and their ability to damage neoplasms (a matter 
of long-standing interest in the Institute stem- 
ming from the early work of Dr. M. J. Shear), 
plus the increasing knowledge of the production 
by the body of a number of substances, including 
antibodies and special enzymes, in response to 
infection and tumors have led to expansion of 
programs in the Laboratory of Chemical Pharma- 
cology in the Polysaccharide Section. 

Endotoxin Studies. Bacterial endotoxins, poly- 
saccharide materials now prepared by Drs. M. 
Landy and E. Ribi (NIAID) in highly potent 
form with no more than 0.5 percent nitrogen and 
1.7 percent fatty acid ester, are highly useful 
materials in the study of immunity, and with 
these and the highly sensitive immunological 
techniques being developed by Dr. Landy and 
his associates, certain phenomena exhibited by 
the whole organism and by cells that were puz- 
zling in the past are now becoming clear. The 
nature of the effects of endotoxin on mammals 
is under study in several laboratories, including 
the various means by which host susceptibility 
can be modified. Although intensive investiga- 
tion is under way on the state of refractoriness 
or tolerance produced by prior treatment of an 
organism with endotoxin, kinds of treatment that 
greatly increase susceptibility are less well de- 
fined. These latter are: reticuloendothelial sys- 
tem-blockade, adrenalectomy, administration of 
BCG vaccine, and tumor implantation. 

In preparation for more extensive work de- 
signed to learn whether these diverse treatments 
produce in the host any change in common, Drs. 
Landy and R. J. Trapani, in collaboration with 
Dr. E. Suter (University of Florida), have in- 
vestigated the possibility of additive or synergis- 
tic effects of a combination treatment of CAFi 
mice with BCG vaccine and implantation with 
sarcoma 37. Results were determined following 
subsequent challenge with Sal. enteritidh endo- 
toxin. No synergistic effect was found ; the effects 
were not additive, and the results indicated inter- 
ference of the action of BCG by the tumor. 

In the course of studies on alterations in the 
somatic endotoxin complex of Gram-negative bac- 
teria that occur during the serum bactericidal 



process (see last year's report), it was observed 
that very small amounts of antibody sufficed for 
bactericidal activity. Dr. Landy, with Drs. J. 
Whitby (guest scientist) and J. Michael (Visit- 
ing Scientist), has developed a simple, specific 
and objective assay for bacterial antibodies that 
is much more sensitive than previously developed 
procedures. Tests with immunologically cali- 
brated antiserum against Sal. typhosa showed 
that the smallest amount of antibody measurable 
by the method was 0.0002 microgram antibody 
and that 0.001 microgram provided marked bac- 
tericidal effect. On this basis normal mouse 
serum was estimated to contain approximately 
0.01 to 0.02 microgram antibody nitrogen (against 
Sal. typhosa) per milliliter. 

In a careful study of the pyrogenic response 
of the rabbit to endotoxin, Drs. Landy and W. 
Keene (PHS Hospital, Boston) have also devel- 
oped a more precise bioassay for endotoxin by 
means of a procedure requiring administration of 
amounts of endotoxin that will produce febrile 
responses that fall on the linear portion of the 
dose response curve. 

The inactivation of endotoxin by blood plasma 
described in previous annual reports was found 
to have characteristics of an enzyme-catalyzed 
reaction. The progress of this reaction was fol- 
lowed by measuring residual substrate (endo- 
toxin) concentration in the pyrogen assay, and 
the assay was based on the assumption that the 
residual fever-producing substance in the plasma- 
endotoxin reaction mixture was unaltered endo- 
toxin. In the previous report, it was suggested 
that the residual pyrogen of incubated plasma- 
endotoxin reaction mixture had the pyrogenic 
properties of endogenous serum pyrogen. With 
Dr. Keene, Dr. Landy has now demonstrated 
that animals tolerant to endotoxin are also toler- 
ant to the residual pyrogen. In addition, daily 
injections of the residual pyrogen induced endo- 
toxin tolerance. Since the most distinctive quality 
of endogenous pyrogen is that it is equally 
pyrogenic for normal and endotoxin-tolerant ani- 
mals, it appears that the residual pyrogen of 
incubated plasma endotoxin reaction mixtures is 
not endogenous pyrogen. 

Not only is endotoxin inactivated by blood 
plasma, but tissues are also capable of inacti- 
vating endotoxin. As in the case of the inacti- 
vating system in blood plasma, the in vitro 



NATIONAL CANCER INSTITUTE 



37 



inactivation of endotoxins by cell-free homogen- 
ates of perfused rabbit liver also appears to be 
an enzyme-catalyzed reaction, but unlike the for- 
mer is not influenced by calcium ions (studies by 
Drs. Landy and Trapani with Dr. V. Warav- 
dekar — AFIP) . Preparations derived from rab- 
bit liver display good endotoxin-degrading ac- 
tivity, but several other tissues from rabbit 
showed extremely low or no activity. Extracted 
cells also yielded clear preparations low in nitro- 
gen and total solids that were potent in destroy- 
ing pyrogenicity and tumor-damaging activity of 
bacterial endotoxin. Rabbit granulocyte homo- 
genates were likewise found to be effective by 
Drs. Landy and A. L. Notkins in activating 
endotoxin, although other formed elements of 
blood, including human erythrocytes, platelets 
and rabbit monocytes were not found to inactivate 
endotoxin. Administration of large amounts of 
endotoxin to mice did not lead to elevation of 
serum lactic acid dehydrogenase, an intracellular 
enzyme that might be expected to be elevated in 
the serum if the endotoxin were producing dam- 
age to granulocytes as has been claimed in re- 
ports in the literature. Furthermore, no release 
of this enzyme was detected when mouse perito- 
nea] macrophages were incubated with endotoxin. 
Drs. M. Woods and Landy have shown that 
the capacities of a series of endotoxins (from 
different Gram-negative genera, isolated by dif- 
ferent procedures, and having different potencies 
in eliciting characteristic effects in the host) cor- 
related well with stimulation of glycolysis in 
vitro. Moreover, experiments on the degradation 
of endotoxin by humoral and intracellular en- 
zymes of the host, or by progressive acid hydroly- 
sis, resulted in a loss of capacity to stimulate 
glycolysis. Both aerobic and anaerobic glycolysis 
are stimulated ; the lowest effective concentration 
of endotoxin is about 0.0001 microgram per ml., 
and 0.3 microgram per ml. generally provides 
maximal effect. Similar effects could be produced 
in vivo as measured with in vitro determinations 
of glycolytic activity of peritoneal macrophages 
obtained from mice given endotoxin a few hours 
previous to the isolation of the cells. 

Tumor Cytotoxic Factors in Serum. Systematic 
investigation of the antibody -complement system 
in normal serum lethal to mouse tumor cells, 
which was reported on last year, has been largely 



completed by Drs. Landy, Trapani, Michael and 
Woods. The general findings are that normal 
sera from many animal species exhibited in vitro 
a lethal effect on ascites cells of sarcoma 37 and 
a considerable number of other tumor cells of 
mice. This in vitro activity of serum on the 
tumor cells of a heterologous species was mani- 
fested by: failure to proliferate in a susceptible 
host; altered permeability to eosin; morphologic 
changes ; and cessation of glycolytic activity. The 
system in serum responsible for these effects con- 
sisted of a natural antibody and complement. 
This antibody had specificity for an antigen 
present in a variety of tumors and lymphoid 
tissues of mice. In continuation of the study, the 
failure of fresh human serum to exert a com- 
parable effect on tumor cells in vivo was shown 
to be due to uptake of the antibody by normal 
lymphoid cells of the mouse. In tests for this 
cytotoxic activity, absence of the cytotoxic effect 
was found in 7 of 17 species studied. Further 
evaluation indicated, however, that the class of 
"inactive" species did in fact possess a functional 
antibody and complement against a bacterial com- 
ponent. Furthermore, sera from two "inactive" 
species, such as hamster and guinea pig, when 
combined would give an active preparation as 
measured by red cell lysis in an appropriate sys- 
tem. It is therefore evident that serum from 
various species may differ strikingly in the prop- 
erties of complement they contain. 

In collaboration with Dr. R. Smith, the levels 
of cytocidal activity (mouse ascites tumor test 
systems) in the serum of cancer patients was 
compared with that in normal human serum by 
Drs. Burk and Woods. The action of such sera 
(active and 52° C.-inactivated) on the patients' 
own cancer cells was also investigated. Prelimi- 
nary results indicate somewhat higher than nor- 
mal levels (2- to 3-fold) of cytocidal activity in 
the sera of cancer patients. 

Antigens Against Human Carcinomas. The 
complexities encountered in the study of host 
humoral and cellular factors in natural immunity 
to infection and neoplastic disease and the criti- 
cally sensitive techniques required for such inves- 
tigations are well illustrated in the experiments 
summarized above. Perhaps for these reasons 
more than any other, progress in immunology and 
cancer has been slow despite a vast amount of 



38 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



effort expended in this direction over the past 
50 years. With such modern procedures, Dr. B. 
Bjorklund (State Bacteriological Institute, Stock- 
holm) has conducted experiments which led to a 
recent report that human carcinomas have anti- 
genic properties different from those of normal 
human tissues. By prolonged immunization of 
horses with preparations of pooled human car- 
cinomas, an antiserum cytotoxic for HeLa cul- 
tures was obtained. This cytotoxicity, which was 
not affected by absorption with normal human 
tissues, was removed by absorption with human 
carcinoma tissue or by HeLa cells. 

Drs. Landy and Trapani, in collaboration with 
Dr. Bjorklund have initiated investigations which 
not only sought to confirm the principal findings 
of Bjorklund, but also were designed to explore 
the possibilities of developing serological proce- 
dures for more extensive work with such anti- 
genic preparations. Their serological experiments 
have indicated that it is possible, under appro- 
priate conditions, to "coat" tannic acid treated 
erythrocytes with preparations extracted from 
HeLa cells with buffer at pH 8. The treated 
erythrocytes were agglutinated to high titer by 
horse antiserum against pooled human carcino- 
mas. It was suspected that the antiserum also 
contained antibodies against normal tissue anti- 
gens. The presence of such antibodies was dem- 
onstrated with the use of tanned erythrocytes 
coated with pooled normal human serum. These 
cells were also agglutinated to high titer by the 
horse antiserum. Practically all of the antibodies 
reactive with human serum coated erythrocytes 
could be removed from the antiserum by absorp- 
tion with cells suspensions of normal human tis- 
sues. However, the reactivity of such absorbed 
serum with HeLa antigen-coated erythrocytes, 
and its cytotoxicity for HeLa cultures, were not 
discernibly affected. On the other hand, analo- 
gous absorption of the antiserum, with Bjork- 
lund's antigen preparations derived from human 
carcinomas, removed cytotoxicity for HeLa cul- 
tures and reactivity for erythrocytes coated with 
HeLa antigen; it is noteworthy that reaction 
with erythrocytes coated with normal human tis- 
sue antigens remained unchanged. The absorp- 
tion technique was controlled by parallel treat- 
ment of an antiserum prepared in rabbits by 
immunization with normal human serum. This 
antihuman serum was cytotoxic for HeLa cells 



and agglutinated erythrocytes coated with HeLa 
antigen or with preparations from normal human 
tissues. However, these properties were no longer 
evident after the serum had been absorbed with 
normal human tissues. 

Immune Tolerance Induced by Breeding. Last 
year, Drs. M. K. Barrett and E. J. Breyere (Re- 
search Fellow, reported the interesting finding 
that a state of immunological tolerance can be 
induced in female mice by breeding with males 
of another strain which makes them suscepti- 
ble to transplants of tumors and skin from the 
strain of the male with which they were bred. 
Further details have been developed, and, to date, 
the effect has been observed in three genetic sys- 
tems with three transplanted tumors and with skin 
homografts in two combinations. The H-2 histo- 
compatibility locus does not appear to be involved. 
This tolerance is highly specific and depends upon 
a genetic concordance between the transplanted 
tissue and the sire of previous litters. Tolerance 
was not found in females parous from intrastrain 
matings, nor in virgins, nor in those parous by 
males of still another strain unrelated to the 
graft. Experiments under way have thus far 
shown the following: (1) Tolerance increases 
with multiparity. (2) It is effective both in the 
case of "natural" and induced resistance. (3) It 
is long lasting, i.e., at least 150-200 days in the 
mouse. (4) A previously induced immune state 
can be partially abrogated by specific parity. 
(5) This tolerant state is resistant to the usual 
immunization procedures, i.e., immunity cannot 
be induced in tolerant females by an inoculation 
of homologous blood and a degree of tolerance 
remains even after the rejection of a homologous 
skin graft. (6) The effect of parity is not due 
to mere exposure to the male in caging, and (7) 
the presence or absence of the mammary tumor 
milk agent in the male had no detectable influence. 

Polysaccharide Investigations. The in vivo 
production of cytoplasmic vacuoles in ascites tu- 
mor cells following the intraperitoneal adminis- 
tration of polysaccharide was reported previously 
by Dr. M. Belkin. The phenomenon, which oc- 
curs only in vivo, appears to be the result of an 
antigen-antibody reaction. In collaboration with 
Dr. Dalton in studies of treated cells of three 



NATIONAL CANCER INSTITUTE 



39 



ascites tumors, the following results have been 
observed with electron microscopy: 

(1). In sarcoma 37 cells the vacuoles ap- 
pear to arise primarily at the site of virus 
particle formation ; but others arise also from 
preformed small vacuoles and "inclusion 
bodies" which are occasionally present. 

(2). Vacuoles develop in Yoshida ascites 
cells preformed small vacuoles and mem- 
brane-enclosed, electron-dense inclusion 
bodies. 

(3). In Ehrlich ascites cells, few pre- 
formed vacuoles are present in untreated 
cells, and the vacuoles develop more slowly 
from membrane-bound, electron-dense "in- 
clusion bodies"; the cells of this variant of 
Ehrlich ascites tumor were rarely found to 
contain virus particles. 
The programs of the Macromolecular Chemis- 
try Section, Laboratory of Chemical Pharmacol- 
ogy, have as their over-all aim study of the 
interaction of biological systems on the macro- 
molecular level, with special emphasis on the 
forces that initiate the interactions and that 
orient the interacting biological systems in a 
manner of high efficiency. Included in these pro- 
grams are studies on polysaccharides, and impor- 
tant information about these and related com- 
pounds complement the results of investigations 
made by Dr. Landy and his group, often obtained 
in collaboration. 

The extensive collection of polysaccharides syn- 
thesized by Dr. P. T. Mora and Mr. J. W. Wood, 
which includes polymers of various sugars and 
their substituted derivatives with negative (acid) 
groups (such as polyglucose sulfate or carboxyl 
derivatives), has been enlarged now to include 
derivatives substituted with positive (basic) 
groups. By synthesizing amino derivatives, they 
have obtained polymers with graded substitution 
of positive groups. In the course of the work, 
new methods were developed which led to the 
introduction of unprecedentedly high amounts of 
amino groups into polysaccharides: (1) The re- 
duction of the tri-Seto-cyanoethyl derivative of 
polyglucose with lithium aluminum hydride in 
tetrahydrofurane, and (2) the reaction of 1-N- 
diethylamino-2, 3-eposypropane with polyglucose 
in aqueous sodium carbonate solution. Some of 
the synthetic polysaccharides (polyrhamnose and 
polymaltose) possess considerable lipemia-clear- 



ing activity without demonstrable toxic or anti- 
coagulant activity, and some others inhibit mito- 
sis in tissue culture (findings of Professor R. 
Meir, Ciba, Ltd., Basle and Dr. A. Di Marco, 
University of Milan, in collaboration with Dr. 
Mora). These synthetic, charged polysaccharide 
derivatives can also be usefully employed in 
fractionation procedures to obtain at a certain 
pH a precipitated complex with a biological 
macromolecule. After centrifugation and sepa- 
ration from other soluble components, the pre- 
cipitate can be easily dissociated again by chang- 
ing the pH or salt concentration. Such a 
procedure is now under study as a means of 
fractionating the endotoxin detoxifying compo- 
nent of Landy. 

Additional information has been obtained on 
the polysaccharide (P-45) prepared from Ser- 
ratia marcescens by a new, large-scale method of 
Mr. A. Perrault reported on last year. This 
polysaccharide endotoxin is an acid polysac- 
charide complex. In earlier work with Dr. E. 
Merler, Dr. Mora found 80-100 negatively 
charged groups with pKa = 2.3 in a 100,000 
molecular weight unit. With Mr. B. G. Young, 
the titration of the negative polysaccharide with 
positively charged macromolecules, such as poly- 
myxin, protamine and lysozyme, has been studied. 
There was a neutralization observed at a pH 
range where the maximum increase in turbidity 
indicated that strong macromolecular interaction 
(precipitation) took place. Generally, with the 
positive macromolecules the tumor necrotizing 
activity decreased, but the pyrogenic activity re- 
mained constant. However, after interaction with 
lysozyme the tumor necrotizing activity slightly 
increased, and after prolonged incubation for one 
or two weeks the fever-producing activity sub- 
stantially decreased. The interaction with lyso- 
zyme was accompanied with changes in the sedi- 
mentation: lower molecular weight components 
appeared. Some fractionation studies were car- 
ried out in the ultracentrifuge in collaboration 
with Dr. W. Carroll (NIAMD) . The P^5 prep- 
aration was also found to be effectively deaggre- 
gated by treatment with the anionic detergent: 
sodium lauryl sulfate. Sedimentation of the 
original P-45 in the ultracentrifuge shows two 
peaks, one S 20 = 4, and another S 20 = 12. After 
treatment with sodium lauryl sulfate, there ap- 



40 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



pears only one peak at S J = 2. However, up to 
now, the sodium lauryl sulfate could not be re- 
moved without causing reaggregation. The bio- 
logical activity of the S J = 2 component was 
unchanged in the presence of the sodium lauryl 
sulfate. This indicates that the biological activ- 
ity is retained in a relatively small molecular 
weight unit of about 10,000 to 20,000. 

These studies are of importance in understand- 
ing the relationship of biological activity of a 
macromolecule and the types, number and distri- 
bution of active sites on the molecule. Studies 
have been initiated to differentiate the enzymatic 
and antigenic sites on an enzymatically active 
protein (RNAase) by electrostatic complexing 
and blocking of certain limited portions of the 
protein surface. Other serum components inter- 
fered with the enzymatic assay when impure 
RNAase-antibody was added to RNAase, and 
purified antibody is now being prepared (method 
of Singer). Under study are influences on en- 
zymatic activity of complexes with such antibody 
and effects of adding negatively-charged macro- 
molecules on enzyme activity, enzyme-antibody 
interaction, and in vivo development of antibodies 
to RNAase. Enzyme-substrate interactions are 
also being investigated by similar approaches. 

Metabolism of the Tumor- Bearing Host 

Body Composition Studies. Drs. J. White and 
F. Miller and Mrs. J. Toal (in collaboration 
with Drs. J. Bloch, A. Harris and N. Berlin) 
continue to add knowledge on the changes in body 
composition and physiology which occur in tu- 
mor-bearing rats. Last year, in a study designed 
to gain information on the increased require- 
ment for sodium ion by rats bearing subcutane- 
ous, progressively growing Walker carcinosar- 
coma 256, it was found that when the tumor was 
more than 10 percent of the body weight pro- 
nounced retention of sodium occurred. Analyses 
of carcasses and tumors have now shown that the 
amount of sodium in the tumor accounted for 
almost all of the total sodium retained. When 
the concentrations of nitrogen and electrolytes in 
carcasses of rats bearing the tumor were ex- 
pressed on a fat-free, wet weight basis, it was 
found that sodium, chloride and nitrogen were 



increased above normal values and potassium and 
water were decreased. Samples of muscle and 
bone showed no increase in sodium and chloride 
ion concentrations, and, although increases were 
observed in skin, they were not enough to ac- 
count for the increases found in total carcass. 
From studies with undernourished mice, it ap- 
pears that, with respect to carcass composition, 
the tumor-bearing rat in positive nitrogen bal- 
ance resembles a nontumor-bearing rat in nega- 
tive nitrogen balance. Analyses of tumor com- 
position show higher concentrations of sodium 
and chloride and lower concentrations of potas- 
sium and nitrogen in necrotic tissue than in vi- 
able tissue. 

Rats bearing the Murphy-Sturm lymphosar- 
coma intramuscularly were also studied. Car- 
casses showed changes similar to those observed 
with the Walker tumor-bearing rats. Total so- 
dium retained by the Murphy-Sturm tumor-bear- 
ing rats, however, was much less than that re- 
tained by the Walker tumor-bearing rats. 

In order to compare the rate of equilibration of 
water in a variety of tumors with water in blood 
and other tissues, Drs. Millar and White injected 
tritiated water intravenously into ether-anesthe- 
tized rats and mice bearing different primary or 
transplanted tumors and obtained samples of car- 
diac blood, tumor and other tissues at subsequent 
times. From radioactivity measurements on the 
water vacuum-distilled from the samples, equili- 
bration values from the tissues were compared 
with those of cardiac blood samples. With the 
exception of mouse ascites tumor, all viable tu- 
mor tissues reached equilibrium with blood in 5- 
10 minutes. Tumor equilibration time for the 
tumors were similar to those for muscle, greater 
than those for liver, but less than those for skin. 
Mouse ascites tumor equilibration times were 
about thirty minutes. The results suggest that 
the major factor in regulating water exchange in 
tumor tissue is the blood supply to the tumor. 
The findings have implications of importance to 
the study of the rates at which drugs enter tumor 
and normal tissues, and antitumor drugs will be 
evaluated. 

Previous work by Drs. R. E. Greenfield, Jr. 
and V. E. Price dealt with the etiology and na- 
ture of anemia of tumor-bearing subjects. These 
studies demonstrated that the anemia of rats with 
certain tumors could be quantitatively correlated 



NATIONAL CANCER INSTITUTE 



41 



with the loss of cells by hemorrhage into the 
area of the tumor. In the course of this work it 
was observed that rats with marked anemia lost 
weight more rapidly, became cachectic and died 
while the tumors were in the range of 20-30 per- 
cent of the body weight, whereas in animals with- 
out anemia the onset of weight loss and cachexia 
was much slower, and the tumors frequently 
reached 50-65 percent of the body weight. How- 
ever, in further study on the relationship of the 
anemia to the cachectic process, it was observed 
that during the period of most rapid tumor 
growth, and despite the onset of anemia, rats 
bearing the Lymphosarcoma R2788 gained ap- 
preciably more weight than the normal controls 
although they both had almost identical food 
consumption. 

Dr. M. Eechcigl, Jr. and Dr. Greenfield have 
done additional experiments designed to eluci- 
date the nature of these findings. Carcass and 
tumor analyses showed that the lymphosarcoma- 
bearing animals had an increased percentage of 
water sufficient to explain their increased weight 
gains. An equally high degree of hydration was 
observed in rats bearing Hepatoma 3683 whose 
body weight gains were lower than those of the 
normal controls. The combined nitrogen content 
of the tumor and the carcass of both groups of 
cancerous rats was similar to or smaller than the 
body nitrogen of the control animals, while the 
fat content of the tumor-bearing rats was mark- 
edly reduced. In confirmation of these findings, 
balance studies showed nearly the same retention 
of nitrogen in rats bearing the Lymphosarcoma 
R2788 as in the controls, but there was a greater 
retention of water and sodium, concomitant with 
the increased weight gains in the tumor-bearing 
animals. 

During the terminal stages, the lymphosar- 
coma-bearing rats were observed to suffer from 
anorexia, loss in total body weight and finally 
died. In this period the animals were in a nega- 
tive nitrogen and potassium balance, and the 
water retention was reduced below the normal 
level while the sodium balance was not appre- 
ciably affected. The positive sodium balance to- 
gether with the decrease in the potassium reten- 
tion suggest the possibility that, to a considerable 
extent, the terminal dehydration of the tumor- 
bearing animals was due to loss of intracellular 
fluid. In these studies, the onset of anorexia and 



the negative nitrogen balance was not prevented 
by increasing the sodium chloride intake of the 
animals, and indeed a decrease in survival time 
was observed among the tumor-bearing animals 
ingesting saline. 

Chemically Defined Diets. Drs. M. Winitz and 
S. M. Birnbaum and Mr. M. C. Otey have ex- 
tended their studies with chemically defined diets 
which were reported on last year. Earlier studies 
have revealed that chemically-defined diets com- 
posed of the 10 essential L-amino acids (in levels 
recommended by Rose et al.), 8 nonessential L- 
amino acids, the requisite vitamins and salts, glu- 
cose and ethyl linoleate, when provided to rats 
ad libitum as 50 percent solutions in water, suf- 
ficed to meet the nutritive requirements for 
growth, lactation and reproduction over several 
generations. In the attempt to develop a diet 
wherein each of the essential L-amino acids was 
present at the minimal level consistent with op- 
timal growth, diets were prepared which incor- 
porated each of the essential amino acids (taken 
one at a time) in an amount ranging from to 
120 percent of that previously employed. By 
variation of the nonessential nitrogen level all 
mixtures were kept isonitrogenous but were other- 
wise identical. Such diets were fed to weanling 
male, Sprague-Dawley rats over a 10-day period, 
and the minimal level of each essential amino 
acid commensurate with optimal growth was as- 
certained. Minimal levels of the essential amino 
acids, relative to the levels in diets previously 
studied, were as follows: L-lysine, 60 percent; 
L-phenylalanie, 40 percent ; L-tryptophan, 80 per- 
cent; L-isoleucine, 100 percent; L-methionine, 60 
percent; L-leucine, 100 percent; L-valine, 80 per- 
cent; L-histidine, 60 percent; L-threonine, 100 
percent. With these ratios as the basis, it became 
possible to formulate chemically-defined diets 
whereon weanling rats grew at a rate of over 4g. 
per day and with more efficient utilization of 
nitrogen than had been observed hitherto. 

It has been known for some time that certain 
D-amino acids could substitute for their optical 
antipodes, some more effectively than others, as 
essential amino acids required for nutrition, pre- 
sumably through successive conversion to the cor- 
responding alpha-keto acid (catalyzed by D- 
amino oxidase) and, through transamination, to 
the L-antipode. Last year studies of this type 



42 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



utilizing chemically-defined diets were conducted 
on methionine, leucine and tryptophan. Although 
the conversion of D-leucine to the corresponding 
alpha-keto acid (catalyzed by kidney D-amino 
acid oxidase) proceeds at an appreciably faster 
rate than the analogous reaction with D-trypto- 
phan in vitro, the degree of utilization of the 
latter amino acid in vivo exceeds that of the 
former one. A comparable situation has now 
been found with D-alloisoleucine, which was re- 
ported in the literature to be completely ineffec- 
tive as a replacement for L-isoleucine in the diet 
of the rat, but which is, at the same time, very 
rapidly oxidized by D-amino acid oxidase in 
vitro. In order to ascertain why the ability of 
the organism to utilize the D-antipodes of the 
essential amino acids varies so considerably, and 
why results are sometimes obtained that are in 
variance with those that would be expected on 
the basis of in vitro enzymatic behavior, experi- 
ments were set up in which groups of animals 
were fed chemically-defined diets wherein the L- 
isoleucine component was replaced, in three sep- 
arate diets, with one, two and three equivalents, 
respectively, of D-alloisoleucine. Although no 
growth was observed with the first two diets, a 
marked growth response was noted with the third. 
The data therefore revealed that D-alloisoleucine 
could be utilized in lieu of L-isoleucine if present 
in sufficiently high concentration in the diet. Ex- 
amination of the urine of these animals also indi- 
cated that the D-alloisoleucine is excreted at an 
appreciably more rapid rate than is D-leucine, 
D-tryptophan or D-methionine and thus pro- 
vided a plausible explanation for both the ability 
of D-alloisoleucine to support growth when pro- 
vided in the diet in relatively high concentration 
and for the relatively lesser degree of utilization 
observed with D-alloisoleucine than with the 
other above-mentioned D-amino acids, despite its 
appreciable susceptibility to D-amino acid oxi- 
dase in vitro. The data indicated that the de- 
gree of utilization of D-amino acids by an animal 
organism is conditioned not only by the rate at 
which they are enzymically converted to their L- 
counterparts in vivo, but also by the extent of 
their renal excretion. 

Previous experiments in the Laboratory of Bio- 
chemistry have indicated that satisfactory nutri- 
tion could be provided for rats on the chemically- 
defined diet offered as 50 percent aqueous solu- 



tions. From controlled experiments conducted 
this past year, it has been shown that the 50 per- 
cent water soluble diet is adequate for normal 
growth and well-being of rats, at least for a 
limited period of time, in the absence of inde- 
pendently supplied water. 

In collaboration with Dr. C. I. Jarowski (Chas. 
Pfizer and Co. ) , Dr. Winitz has begun an evalua- 
tion of the concept that the free amino acid levels 
in the blood plasma of a given animal species, in 
the fasting state, might serve as a precise indi- 
cator of the dietary amino acid requirements of 
that species. Studies were done to compare the 
growth response of rats provided with the most 
efficient water-soluble, chemically defined diet 
hitherto devised in the Laboratory of Biochem- 
istry, with that of rats fed comparable diets 
wherein the essential amino acids were present 
in the same relative ratios as they appeared in the 
plasma of the fasting rat. The data demon- 
strated that the former and latter diets elicited 
comparable growth rates, despite striking differ- 
ences in their essential amino acid compositions. 
Thus, in the rat, the concept appears valid, and, 
if, after work in other species, the principle be- 
comes established, it would be of considerable 
assistance in the formulation of diets. 

In Vrvo Perfusion Studies. In efforts designed 
to find a means for growing a tumor isolated 
from the surrounding tissues and connected with 
the host only by one artery and one vein, Dr. P. 
Gullino (Visiting Scientist), with Mrs. F. H. 
Grantham, has developed a technique for grow- 
ing tumors in pouches developed in kidneys (or 
in ovaries). A kidney was isolated from the sur- 
rounding adipose tissue with the vascular pe- 
duncle as the only connection with the host. A 
tumor was then transplanted into the renal par- 
enchyma, and the kidney enveloped in a bag of 
paraffin was placed into the subcutaneous tissue. 
The tumor grew in this bag and destroyed the 
kidney tissue; the renal vessels remained as the 
only connection between the tumor and the host 
(rats, hamsters or mice). 

In rats, renal implants grew up to the size of 
25 grams (about 25 times the kidney's original 
weight) and ovarian implants grew up to 14 
grams (about 400 times larger than the host 
ovary). Two transplants of the same tumor, one 
grown in a paraffin bag and the other grown sub- 



NATIONAL CANCER INSTITUTE 



43 



cutaneously, showed the same correlation between 
wet weight, dry weight and total nitrogen and 
also the same level of anaerobic glycolysis. Ani- 
mals bearing the "isolated" transplant behaved in 
the same manner as those with subcutaneous tu- 
mors as to their change in body weight, food con- 
sumption and water intake. Liver catalase was 
also equally depressed. 

Utilizing this technique, Dr. Gullino, with Mrs. 
Grantham, has measured blood flow from im- 
planted tumors and has studied the effects of cer- 
tain drugs on this blood flow. In an adult rat 
the rate for the ovary and oviduct was deter- 
mined to be about 12 ml. per hour. The same 
amount of blood flows out of tumors weighing 
6.0 grams or more, namely 200 times heavier 
than the ovary into which they were implanted. 
The growth of a tumor implanted in a kidney, 
which has a blood supply 20 times larger than 
the ovary, produces a decrease of the total blood 
flow to the degree that when the tumor has de- 
stroyed all the kidney parenchyma, the blood 
reaches values of the magnitude of those found 
in ovarian implants. Despite a difference of 20 
fold in the blood supply between the ovary and 
the kidney, equal amounts of tumor implanted 
simultaneously in both organs of the same rat 
grew at the same rate, suggesting that a large 
blood supply does not increase the growth rate of 
rat tumors. Adrenalin reduces the blood outflow 
of a tumor and the removal of the coeliac gang- 
lion has the same effect. Choline increases the 
blood outflow in some instances. 

Collagen, which was determined in collabora- 
tion with Mr. H. Taylor by measuring the 
amount of hydroxyproline content of acid hy- 
drolysates of tumors, increases with the growth 
of the tumor. Preliminary comparative studies 
of some enzymatic activities in the efferent and 
afferent blood of the tumor are being done. 

Kinetic Studies in the Tumor-Bearing Ani- 
mals. Last year Drs. M. Eechcigl, Jr. and V. E. 
Price reported on a method for determining the 
kinetics of in vivo synthesis and destruction of 
the liver enzyme, catalase, by use of 3-amino-l, 
2, 4-triazole. With Dr. R. Hartley, they have 
evaluated the validity of this method by an al- 
ternative one in which catalase synthesis was 
blocked by the use of allylisopropylacetamide 
administered intraperitoneally to rats. When 



synthesis was blocked, activity of the remaining 
catalase disappeared with a first order constant, 
K d , of 0.023, corresponding to a rate of disappear- 
ance of 2.3 percent per hour, a figure almost 
identical to that obtained by the older method. 

These methods have been employed in compara- 
tive studies with rats on a protein-free diet, on 
a starvation diet, or bearing a tumor. In the 
liver of starved animals, synthesis and destruc- 
tion proceeded at essentially the same rate per 
gram of tissue as in normal animals for the first 
5 or 6 days of starvation. The liver and total 
liver nitrogen is continually decreasing in size 
during this period, however, so that there is actu- 
ally less catalase being synthesized and destroyed 
in the total liver. In contrast to the picture seen 
in starvation, animals on a protein-free diet of 
carbohydrate, fat, minerals, vitamins and a source 
of fiber showed a decreased rate of catalase syn- 
thesis per gram of tissue or per mg. of nitrogen. 
Although there was less catalase being synthe- 
sized, the fraction of catalase molecules being de- 
stroyed per unit time was within normal limits, 
thus resulting in a lower concentration of catalase 
within the liver. However, in the animals on the 
protein-free diet, the liver size and total liver 
nitrogen decreased only slightly. 

The effects of tumors are more nearly dupli- 
cated by the protein-free diet, for in the tumor- 
bearing animals there is a marked fall in the 
catalase activity per gram of liver, and the liver 
is frequently somewhat enlarged. In both these 
states the total liver catalase is markedly reduced 
as it is in starvation, but it is not accompanied 
by the marked decrease in liver size or total liver 
nitrogen seen in starvation. 

Enzyme Activities of the Tumor-Bearing Host. 
Comparative studies of catalase activity of vari- 
ous hepatomas and of their effects on host liver 
and kidney catalase activity levels have also 
been made by Drs. Rechcigl and Price, in col- 
laboration with Drs. H. P. Morris and H. Sidran- 
sky. A fairly high catalase activity was found 
in the 5123 hepatoma and in the primary and the 
transplanted ethionine-induced hepatomas of 
mice. Almost no activity could be demonstrated 
in the Dunning, Novikoff and the 3683 hepatomas 
of rats. 

Despite the fact that fairly high levels of 
catalase were found in the tumor, the catalase 



44 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



activity of the liver and kidneys of animals bear- 
ing 5123 and ethionine-induced hepatomas was 
depressed to a degree comparable with the other 
tumor-bearing animals tested. The amount of 
depression was correlated with the size of the 
tumor and the loss of the carcass weight. 

In view of the lowering of the liver catalase of 
normal animals following the feeding of a pro- 
tein-free diet, it was of interest to find out 
whether a comparable decrease in the enzyme ac- 
tivity would occur in hepatoma 5123. The ex- 
periments have shown that a protein-free diet 
produces no significant lowering of catalase in 
the 5123 hepatoma, even though the catalase of 
the liver was markedly reduced. 

In spite of numerous reports that a substance 
isolatable from tumor tissue (toxohormone) 
causes reduction of liver catalase activity by 
liberation of the substance into the blood stream 
with local action on the liver catalase, other in- 
terpretations of the phenomena may well deserve 
equal or better support. The reduction in cata- 
lase activity observed in bled animals and in 
those placed on protein-free diet {vide supra) 
suggest other mechanisms, such as a relative nu- 
tritional deficiency or an alteration of the homeo- 
static balance brought about by competing altered 
metabolic pathways. The kinetic models de- 
signed to permit the simultaneous measurement 
of catalase synthesis and destruction, especially 
if confidence in their validity can be maintained, 
for nonsteady states present in tumor-bearing ani- 
mals as more work is done, are likely to aid in 
the clarification of this well-known effect on the 
host by a tumor. 

Transplantation of a primary ethionine-in- 
duced hepatoma resulted in tumors having two 
different levels of catalase activity, one of which 
was twice that of normal liver, whereas the other 
was about half the normal level. Enzyme level 
estimations in tumors of later transplant genera- 
tions of the two lines will be of interest. 

Utsugi has recently reported that hypophy- 
sectomy will prevent the depression of liver cata- 
lase activity. Drs. Rechcigl and Price, with Dr. 
S. Wollman, however, found a marked fall in 
the concentration of liver catalase in hypophy- 
sectomized animals bearing transplanted Woll- 
man thyroid tumors. In fact, the effects of the 
tumor growth and removal of the pituitary gland 
were additive. Starvation is often referred to 



as pseudohypophysectomy, since the effects of 
starvation and extirpation of the gland are simi- 
lar. The similarity was also seen in experiments 
involving liver catalase measurements in hypo- 
physectomized rats, since the livers were only 
about one-third the normal size, and the catalase 
activity per gram of liver tissue was at normal 
or slightly elevated levels. 

The association of elevated serum aldolase and 
lactic acid dehydrogenase with certain tumors 
and with certain lesions of muscle has been well- 
documented in both human and animal subjects. 
Recently, Riley has reported marked elevation of 
serum lactic acid dehydrogenase in mice that had 
received injections of cell-free extracts. Drs. 
Greenfield and R. Berry have confirmed these 
findings in mice; however, rats injected with a 
large series of transplanted tumors had no such 
elevation, and cell-free extracts of tumors were 
also without effect in these animals. Preliminary 
studies suggest that the elevation of lactic acid 
dehydrogenase as seen in mice is not due to a 
viral agent since elevation was still obtained fol- 
lowing administration of cell-free extracts ir- 
radiated with a dose sufficient to inactivate most 
viruses. 

Deoxyribosides of the Tumor-Bearing Host. 
Investigation of the deoxyribosides of rat tissue 
and the metabolism of these compounds during 
liver regeneration and growth is under way by 
Dr. J. Rotherham. Deoxycytidine, 5-methylde- 
oxycytidine, deoxyuridine, and unidentified de- 
oxynucleotides were found in the urine of normal 
rats fed a casein diet. When rats are maintained 
for 2 or 3 weeks on a diet which contains no 
source of preformed deoxyribosyl compounds, 
these compounds are still continuously excreted 
in the urine, an indication that they are formed 
endogenously (bacterial action has not been ruled 
out). Studies with this special diet indicated 
that the level of excretion fell in hepatectomized 
animals on the second day after the operation. A 
fall in levels of excretion occurred in chow fed 
animals bearing the Novikoff hepatoma when 
the tumor was growing rapidly, but a similar fall 
was observed in pair-fed control animals. 

Urinary Excretion Patterns of Nucxeto Acid 
Congeners in Leukemia Patients. Further 
progress continues to be made by Dr. J. Reid 



NATIONAL CANCER INSTITUTE 



45 



and Mr. W. Bell in the difficult and complex in- 
vestigation of the urinary excretion patterns of 
nucleic acid congeners and other substances in 
leukemia and normal human objects. The meth- 
ods used are primarily chromatographic with 
employment of ultraviolet absorption spectra de- 
terminations of the eluate fractions; machine 
methods of data processing are being introduced. 
Dietary composition exerts an important effect 
on the normal urinary pattern. The absorption 
curves were changed in all chromatographic frac- 
tions when a freely selected diet was given com- 
pared with the results seen when a low-purine, 
low-ribose control diet was administered. Pat- 
terns seen in urines from two untreated patients 
with acute myeloblastic leukemia and from two 
untreated patients with acute lymphoblastic leu- 
kemia differ from those seen in urine from nor- 
mal subjects. 

Vector Analysis-Computer Techniques. In a 
study designed to test the feasibility of analyzing 
multicomponent mixtures (by means of vector 
analysis-digital computer techniques applied to 
the ultraviolet absorption spectral curves of 
chromatographic eluates of ribonucleic acid 
(ENA) hydrolysates), Drs. Reid and A. Pratt 
have made considerable progress. With RNA 
as a model, it is intended to develop the tech- 
niques to a point where they can be used to ana- 
lyze urinary excretion patterns from leukemia 
patients. RNA hydrolysates consisting of the 
four major nucleotides and three other compon- 
ents present in smaller amounts have been ana- 
lyzed by the computational method with a pre- 
cision equal to or better than that of standard 
methods and an accuracy about as good. When 
the spectra of the minor components have been 
determined, the accuracy will be increased. With 
the aid of Mr. N. Coffey, automation of the sys- 
tem is being accomplished, thus permitting fu- 
ture practical application of the approach to 
large numbers of samples. 

Energy Expenditure Studies. Further improve- 
ments in mathematical formulation and instru- 
mentation utilized in direct and indirect calori- 
metry studies have been made by Dr. Pratt and 
Mr. W. White. Particular attention is given to 
methods because of the need for high precision 
in evaluating the independent measures of energy 



expenditures (direct and indirect) of the tumor- 
bearing and normal animal. From mathematical 
approaches developed by Dr. Pratt to the solu- 
tion of the dynamic water pool determination 
and the determination of the body volume of an 
animal in the respiratory chamber, it appears 
possible with the use of tritium-labeled water to 
obtain samples of respiratory water without dis- 
turbing the animal and, if measurements can be 
made with sufficient accuracy, to estimate the 
total body water and body volume of the animal 
in the chamber. By use of density measurements 
of the dry total carcass and of bomb colorimetric 
measurements of the calories per gram of dry 
total carcass, Dr. Pratt is now able to calculate 
from relationships he has formulated, the nonfat 
and fat fractions without the necessity of chemi- 
cal separation. These new approaches provide 
additional means for evaluating independent 
measures of energy expenditures, one of the im- 
portant areas of investigation of metabolism of 
the tumor-bearing subject. 

With Drs. H. Benzinger and C. Kitzinger 
(Naval Medical Research Institute, Bethesda, 
Md.), Dr. Pratt is developing techniques of clin- 
ical thermometry to ascertain body temperature- 
energy metabolism relationships. A basic rela- 
tionship between skin temperature, intracranial 
temperature and energy production has been 
found in studies on human subjects. The meth- 
ods developed should also prove useful in other 
types of clinical studies. 

Tumors and Their Properties 

Staff members of the Institute maintain about 
200 transplantable neoplasms. This very valu- 
able resource includes many tumors of special 
characteristics and is a vital part of the arma- 
mentarium of cancer investigators, not only with- 
in the Institute, but throughout the world. 

As part of a long-term study of the effect of 
age on rats of six of the NIH inbred strains, 
Dr. K. Snell reports differences among them as 
to the most frequent sites of spontaneous tumor 
formation: Osborne — Mendel, adrenal cortex; 
Buffalo, anterior pituitary gland and adrenal cor- 
tex; AXC, testis and anterior pituitary gland; 
Fischer, testis; M520, adrenal medulla; and Wis- 
tar, anterior pituitary gland and mammary gland. 

Acetic acid-soluble collagen in hamsters, which 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



is present in lower amounts in back skin as com- 
pared with abdominal skin, has been found by 
Dr. W. Banfield to decrease in skin with increas- 
ing age except for a rise in the amount from 
abdominal skin between the ages of 28 and 42 
days. In the process of maintaining old hamsters 
while developing this useful tool for study of 
collagen metabolism, Dr. Banfield has also estab- 
lished 16 transplantable hamster tumors (among 
which are 2 melanomas, 1 adrenal cortical tu- 
mor, 1 myxoma, 1 epidermoid carcinoma, 1 ade- 
nocarcinoma of the bowel and 3 reticulum cell 
sarcomas). 

Miss D. Uphoff is attempting to establish a 
line of mice, C57BL/10D, that develop multiple 
polyposis of the colon. Although polyps are oc- 
casionally found, malignancies have not been 
observed. 

The biologic behavior of the adrenal cortical 
carcinoma 494 (i.e., rats bearing the tumor devel- 
op polyuria, polydipsia, degenerative changes in 
the kidney, and atrophy of adrenal glands, sex 
organs, lymphatic tissue, and pituitary glands) 
has remained the same through 15 transplant 
generations in studies by Dr. Snell. In a col- 
laborative study, Dr. Johnson (NIAMD) has 
found that desoxycorticosterone is the major 
steroid elaborated by the tumor, even though this 
steroid is absent or nearly so in the adrenal 
glands of Osborne-Mendel and other strains of 
rats. 

In an attempt to learn of differences between 
steroid secretions by another adrenocortical car- 
cinoma and normal adrenal cortical tissue of the 
rate and to obtain additional understanding of 
metabolic pathways related to tumor-normal tis- 
sue comparisons, Drs. A. Mulay and E. Price, Jr. 
have found that rats bearing the tumor show 
atrophy of the zona f asciculata and zona reticu- 
laris of the adrenal crotex, a sharp reduction in 
urinary sodium excretion (both in intact and 
adrenalectomized rats), increased potassium ex- 
cretion, hypernatremia, and hypokalemia. Since 
studies on stress in tumor-bearing rats demon- 
strated that the tumor could protect the animals 
against intoxication produced by intraperitoneal 
potassium chloride injection, the tumor appar- 
ently elaborates mineralcorticoid secretions. Pre- 
liminary findings suggest no production of gluco- 
corticoid secretion by the tumor. 

Dr. S. Wollman, as part of a continuing study 



on properties of transplantable rat thyroid tu- 
mors, has found that one tumor line can maintain 
a concentration of radioiodine elevated above 
that of blood and nearly as high as that in nor- 
mal thyroid glands, although this tumor cannot 
form appreciable protein-bound I 131 . Impair- 
ment in transport of radioiodide from blood to 
tumor has also been observed. Dr. J. Tijo 
(NIAMD) has examined the chromosome com- 
plement of three lines of thyroid tumors main- 
tained by Dr. Wollman and found that two 
anaplastic lines had deletions of 4 and 7 chromo- 
somes, respectively, whereas a functional line had 
a deletion of only 1 chromosome. 

Cells from a number of spontaneous and in- 
duced mouse tumors have been injected intra- 
venously by Drs. R. Malmgren and E. Chu, and 
comparisons were made of the nuclear DNA con- 
tent between the initial tumor and the developing 
metastases. There is a tendency for the metastatic 
tumor cells to have a higher nuclear DNA than 
the primary tumor from which they are derived; 
however, it appears that the high ploidy cells 
are not the only ones that are capable of estab- 
lishing metastatic foci. 

Mouse tumors and normal tissues and extracts 
prepared therefrom when injected into mice were 
observed by Dr. Malmgren to increase the mitotic 
activity of liver under certain conditions. The 
factor appears to be heat stable and can be stored 
in vacuo. Heated normal tissue produces an in- 
crease in mitotic activity, but unheated normal 
tissue appears to contain an inhibitor, since it 
can block the effect of tumor tissue. 

With Dr. E. McLaughlin, Dr. Malmgren has 
observed an increase in liver mitotic activity in 
mice and rats that received serum from cancer 
patients. The effect was not seen when serum 
from normal human subjects was given. Using 
tritum-labeled thymidine and isolating DNA 
from the livers at 72 hours, Drs. Malmgren and 
McLaughlin observed an increase in new DNA 
in the livers of partially hepatectomized rats 
treated with serum from cancer patients com- 
pared with rats treated with normal human 
serum. 

Dr. Malmgren found no significant difference 
between the various cells studied from carcinoma 
in situ and invasive cancer patients with tech- 
niques of microspectrophotometry, interference 
microscopy, and planimetry while he was work- 



NATIONAL CANCER INSTITUTE 



47 



ing in Prof. T. Caspersson's Laboratory in 
Stockholm. 

In continuing investigations on cytochemical 
changes in intact living individual cells, Drs. G. 
Williams and A. Peacock have made further im- 
provements in instrumentation used for ultra- 
violet-television microscopy and time-lapse cine- 
matography. By use of this system, intact cell 
functioning can be studied with brief exposure 
of low-level UV radiation, and they have com- 
pleted a study of a series of tetrazolium salts 
utilized to investigate the reaction rates of an 
enzyme system catalyzing the reduction of tetra- 
zolium to formazan and to obtain information 
on the cellular localization of the reaction. In 
liver cells, progressive reduction occurs in the 
vicinity of mitochondria without appearance of 
formazan outside the cell or inside near the cell 
surface. Tetrazolium appears to be freely per- 
meable, and all but 20 percent bound to cellular 
protein can be easily removed with a single wash- 
ing of the cells. Improvements in instrumenta- 
tion include new lenses that permit excellent 
resolution with UV light at 265 m/i in enlarge- 
ments at a magnification of about 30,000 diame- 
ters and a new electronic shutter with precise 
opening times from 5 to 75 milliseconds, or 10 
millisecond intervals at 5, 10, 15, or 30 cycles or 
2- or 3-minute cycles. Exposure of the cells to 
UV light with this TV arrangement is about 
l/100th that required to photograph the activity 
directly on film. With 10 millisecond exposures 
at intervals of 15 seconds, human leukocytes tol- 
erate UV light at 265 m/i for 15 to 30 minutes 
before showing structural and dynamic changes 
of damage. 

Plasma Cell Tumors 

Dr. M. Potter has made additional progress in 
his investigations on plasma cell neoplasms. He 
has found that BALB/c mice given single intra- 
peritoneal injections of adjuvant-staphylococcus 
mixtures, develop plasma cell neoplasms in the 
lipogranulomatous tissue that formed in all mice 
receiving the mixture. Thirty-one plasma cell 
tumors have been found thus far, none developing 
prior to 5 months after injection. Because of the 
complexity of composition of the adjuvant mix- 
ture, studies are under way to determine if single 
components and other materials will also produce 
these neoplasms. To date, 4 plasma cell neo- 



plasms have developed following the administra- 
tion of paraffin oil (Bayol F) alone to 40 mice. 
No plasma cell tumors have appeared to date in 
similar studies in three other strains of mice. 

Growing transplants of plasma cell tumors pro- 
duce quantities of serum myeloma globulin in new 
recipients. Specific myeloma globulins are pro- 
duced by different tumors. When a tumor is 
established in transplant, the specific myeloma 
globulin production is constant for that tumor 
and remains qualitatively the same throughout 
the transplant history. Studies on different pro- 
tein-producing tumor lines derived from a single 
host would be of interest. One case so far has 
been found: the primary host of Adj-PC6 gave 
rise to two different, stable transplant lines, 
Adj-PC6A and Adj-PC6C. One difference in 
the behavior of these two cell lines results in 
precipitation of the myeloma globulins when 
whole serum of tumor-bearing animals is diluted 
in low ionic strength media in the case of the 
former, but not the latter cell line. Of the 24 
different plasma cell neoplasms (20 are of strain 
BALB/c origin), two additional cell lines give 
this precipitation phenomenon; in such cases iso- 
lation of the globulins is simplified. 

The primary peritoneal plasma cell neoplasm 
appears to rise multifocally, for the reactive 
mesentery contains numerous nodules of seem- 
ingly incipient plasma cell tumors. Dr. Potter 
is using an ingenious method to study the mech- 
anisms of their development by transplantation 
of wedges of mesentery containing separated 
nodules into recipient mice. By this method he 
hopes to localize the time and site of origin of 
the carcinogenic event and to obtain discrete tu- 
mors from which lines can be established for 
additional immunological and chemical charac- 
terization of the tumor products. 

Several immunochemical and cytochemical 
studies are under way with Dr. E. L. Kuff. The 
microsomes contain material antigenically related 
to the serum protein myeloma globulins. Ribonu- 
cleoprotein (RNP) particles prepared from mi- 
crosomes by deoxycholate treatment also carry 
the antigenic groups of the myeloma proteins. 
Similar antigenic determinants are found on nor- 
mal immunoglobulins such as gamma-2-globulin 
(SI) and a certain beta-globulin (SII). Three 
antimicrosome antibodes have been studied : 



48 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Tumor source 

MPC 1 

MPC 2 

Adj-PC 5 



Precipitates formed icith 
normal serum 



sn 

SI, SII 
SI. 



Myeloma globulins are related antigenically to 
SI or SII, or very rarely to both. The normal 
SI and SII proteins contain different antigenic 
determinants. Myeloma globulins may contain 
different antigenic determinants, or may lack 
them. Characterization of the myeloma globulins 
of BALB/c mice will be done, and the degree 
of genetic control obtainable in such a study will 
be far superior to that possible with myeloma 
globulins sampled at random from heterogeneous 
populations. 

Dr. M. Landy and Dr. Potter have sought to 
determine whether the abnormally high levels of 
beta and gamma globulins in the sera of mice 
bearing plasma cell tumors (induced with paraffin 
oil and bacteria) represented antibody or dis- 
oriented protein. The serum of mice normally 
contains, in low titer, a broad spectrum of indi- 
vidually specific antibodies to Gram-negative bac- 
terial species. As far as can be determined, these 
antibodies are the result of antigenic stimuli 
supplied by continuous contact of those animals 
with Gram-negative bacteria. It was reasoned 
that in the presence of this persistent, multiple, 
natural antigenic stimulus, the greatly increased 
synthesis of serum globulins, usually associated 
with antibodies, might be reflected in markedly 
increased levels of antibody to these bacterial 
species. Employing the sensitive and objective 
bactericidal assay for antibodies to Salmonella, 
it was found that sera of mice with these plasma 
cell neoplasms contained no greater amount of 
Salmonella antibody than that present in normal 
mice. Since the increased level of globulins did 
not reflect enhanced antibody response to bacteria, 
the function, if any, of globulins is still obscure. 

Using microincineration and Brachet's pyronin- 
methylene green-ribonuclease techniques, Drs. 
MacCardle and Potter have found that gamma- 
globulin secreting plasma cell tumors are gener- 
ally rich in cytoplasmic RNA and in calcium and 
magnesium, whereas those secreting alpha- and 
beta-globulin are poor in cytoplasmic RNA and 
rich in intranuclear RNA. The calcium and 
magnesium content of the latter varies. The nu- 



cleolus of the gamma-globulin tumors is large and 
rich in silica. 

In collaboration with Drs. Potter and Ruth 
Merwin, Dr. A. J. Dalton has examined by means 
of electron microscopy 17 primary and trans- 
planted plasma cell tumors and controls consist- 
ing of normal stimulated and unstimulated 
plasma cells or fibrosarcomas. Maintenance of 
ultrastructural features characteristic of the 
plasma cells is related to retention of function 
(determined by the pattern of specific myeloma 
globulins elaborated by a particular cell type). 
The evidence obtained also suggests that particles 
present in the cytoplasm of cells of all the 
plasma cell neoplasms examined (but not in nor- 
mal plasma cells nor in the cells of other neo- 
plasms) are not the morphologic representation 
of an oncogenic agent responsible for the develop- 
ment of the tumors. 

Dr. H. Kobayashi (Visiting Scientist) with 
Drs. Thelma Dunn and Potter, has demonstrated 
both osteolytic and osteoplastic reactions by 
plasma cell neoplasms using techniques involving 
intravenous injection of the ascites form of tumor 
(growth within the bone marrow resulted) , trans- 
plantation of solid tumor tissue into marrow 
cavities and onto peritoneal surfaces, the use of 
papain-cleared skeletons, and examination of his- 
tologic sections. 

Diffusion Chamber Studies 

The occurrence of several plasma cell tumors 
and sarcomas in mice following the placement in 
the peritoneal cavity of millipore filter diffusion 
chambers was reported previously by Dr. Ruth 
Merwin. In further work with Dr. Thelma Dunn, 
she has found that both tumor types are induced 
more frequently in the BALB/c than in the 
C3H strain and that the presence of tissue inside 
the chamber, stilbestrol infection, and anyone of 
the chamber parts (membrane, lucite or adhesive) 
do not appear to be essential to induction of the 
plasma cell tumors. BALB/c mice react by pro- 
ducing intraperitoneal plasma cell neoplasms 
which are transplantable and have characteristic 
protein electrophoretic patterns resembling those 
of human multiple myeloma. The C3H mouse, 
however, has not produced these neoplasms, but 
severe fibrosis and occasional ossification occur. 

Dr. Emma Shelton has continued her study of 
the behavior of normal and malignant cells 



NATIONAL CANCER INSTITUTE 



49 



grown in double diffusion chambers placed in the 
peritoneal cavities of mice. Fluids, but not cells, 
may pass freely through the membranes of the 
chambers. Growing in the chambers, lympho- 
cytes that were procured from the thoracic duct 
and lymph nodes behaved differently from those 
procured from the peritoneal cavity, indicating 
different potentialities for survival, growth and 
differentiation. The latter formed organized 
fibroblastic sheets and became a minor component 
in the cell population; the former produced few 
sheets and persisted as major components in the 
population. Peritoneal lymphocytes grown in the 
chambers on different physical forms of substrate 
membranes varied in their capacity to form or- 
ganized tissue and failed to undergo the trans- 
formations observed when grown on Millipore 
substrate membranes, except when they grew in 
the fibrous coagulum that sometimes formed. No 
differences were seen when either basic or acid 
ion-exchange resins were included in the cham- 
bers. Preliminary findings with chemical tech- 
niques show an increase in mucopolysaccharide 
correlated with connective tissue growth. Im- 
munological techniques demonstrated passage of 
antibody into the chambers. These different re- 
sponses of cells obtained from different body 
sites may be of importance in determining the 
factors responsible for establishment or lack of 
establishment of metastatic lesions in different 
tissue sites in the body. 

In collaboration with Dr. Virginia J. Evans, 
Dr. Shelton has found that mouse connective tis- 
sue grown for at least a year in diffusion cham- 
bers (through 10 to 12 chamber transplant gen- 
erations) did not acquire malignant properties 
when evaluated by the technique of intraocular 
transplantation. 

Dr. R. T. Bradley (last year an Anna Fuller 
Fund Fellow) and Dr. L. Law are continuing 
studies with a technique that allows one to per- 
fuse substances into the extracellular fluid en- 
vironments of a designated piece of tissue or 
population of cells within normal unrestrained 
experimental animals for periods up to several 
weeks. One study has utilized this procedure to 
perfuse substances continuously into intraperi- 
toneally implanted Millipore filter diffusion 
chambers containing P-388 or L1210 cells. When 
such chambers were perfused with Eagle's basal 
medium (with pyruvate, serine and glutamine) 



for 7 days at the rate of 1 ml. per day, the ap- 
proximate generation time was 12 hours, com- 
pared with 24 to 32 hours in control nonperfused 
chambers. Other perfused media gave poorer 
growth rates, and the effects of various substances 
are under study. 

Tissue Culture Studies 

The careful technique sustained over the years 
in the Tissue Culture Section has permitted the 
cell cultures to be carried aseptically, with an 
absolute minimal and usually no use of antibiotics. 
Recent studies have shown that all cultures car- 
ried in the section are apparently free from pleu- 
ropneumonia and diphtheroid organisms which 
have complicated the interpretation and evalua- 
tion of nutritional, biochemical and other biologic 
data from laboratories relying on antibiotics. 

Comparative studies of normal and malignant 
cells can be facilitated if additional techniques 
are developed that will enable cell strains, already 
adapted to static cultures in chemically defined 
media, to grow luxuriantly in fluid suspension 
cultures without addition of serum or other ill- 
defined components. Agitated culture techniques 
produce stress on the cells grown in fluid suspen- 
sion which varies considerably with the conditions 
employed. Messrs. J. C. Bryant and E. L. Schill- 
ing and Drs. V. J. Evans and W. Earle are in- 
vestigating a number of factors that appear 
important in growing cells in agitated suspension, 
such as the shape and size of the containers, the 
type of surface of the containers, the rate of 
agitation and the presence or absence of certain 
components in the medium, particularly polymers 
of known composition that might play a beneficial 
role similar to that of serum. Last year it was 
reported that addition of methylcellulose to the 
chemically defined medium NCTC 109 produced 
a protective effect on cells grown in agitated cul- 
ture. Higher concentrations of the polymer than 
used last year have led to increased proliferation 
rates of monkey kidney cells. Additions of sper- 
mine or much higher concentrations of folic acid 
than ordinarily employed did not give improved 
results (from reports in the literature, spermine 
has been shown to influence permeability of bac- 
terial cell walls by preventing osmoticlysis and 
high concentrations of folic acid appear to be 
required for certain kidney cell lines grown in 
static culture). In roller tubes, different rates 



50 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of tube rotation (from 19 to 2,400 rph.) have 
been investigated; 1,200 and 600 rph. gave higher 
proliferation rates than those at 2,400 rph. or 
300 rph. or less. 

Further improvements have been made in tech- 
niques utilized in growth of mammalian cells in 
rapidly agitated fluid suspension cultures grown 
in shaker flasks by Messrs. Bryant and Schilling 
and Dr. Earle. By reduction of the size of the 
individual culture flasks, the entire agitation as- 
sembly can be placed within the carefully con- 
trolled temperature room, and manipulation of 
the cultures is greatly facilitated since the fluids 
can now be changed without transfer of the cells 
from the flasks. This new procedure now allows 
rapid proliferation of monkey kidney epithelial 
cells in a protein-free, chemically defined medium 
for the first time. 

Dr. Evans is attempting to adapt freshly iso- 
lated cells of monkey kidney, mouse kidney and 
mouse fibroblasts to grow in a protein-free, chemi- 
cally-defined medium NCTC 109. Maintenance 
of cells and some indication of growth has taken 
place, but it is too early to consider that cell lines 
have become established. Establishment of such 
lines in protein-free medium would be of impor- 
tance to virology studies since the problems en- 
countered with serum-containing media could be 
avoided. Furthermore, pleuropneumonia-like or- 
ganisms would be excluded since the medium does 
not propagate these organisms. 

In collaboration with members of the staff at 
the Navy Tissue Bank (National Naval Medical 
Center, Bethsda) , Dr. Evans has also successfully 
cultured human skin epithelial cells in protein- 
free medium NCTC 109 in both static and agi- 
tated cultures. 

Dr. Earle and Mr. W. T. McQuilkin are at- 
tempting to establish the cinemicrographic tech- 
niques to permit accurate characterization of cells 
in culture, including growth rates, cytokinetic 
periods, intermitotic intervals, and migration 
rates. Considerable variability of behavior among 
cells of pure line populations makes progress 
toward standardization of technique slow. Even 
with preliminary findings, however, it is evident 
that a cell line (NCTC 929-strain L) returned to 
Serum-containing medium after an extended so- 
journ in a protein-free, chemically-defined me- 
dium (NCTC-109) reverts to a state exhibiting 
.characteristics seen in the original strain, but not 



seen while grown in the protein-free medium. 
Thus, whatever the phenomena of adaptation to 
the chemically defined medium might be, the ob- 
served changes are not necessarily permanent. It 
may be recalled that last year's report indicated 
the changes seen when cells of this clone line 
were adapted to growth in a protein-free medium : 
the average generation time was prolonged; the 
migration rate on the glass surface was greatly 
increased; the cells became less compact, refrac- 
tile and granular and rounded up only in 
prophase. 

Transformation Studies. During the past few 
years Dr. Katherine K. Sanford and her associ- 
ates in the Tissue Culture Section have studied 
many types of transformation — neoplastic, en- 
zymatic, morphologic, and chromosomal — occur- 
ring within clones of mammalian tissue cells 
grown in long-term culture. With the identifica- 
tion of several biochemical markers, such as those 
determined by Dr. B. B. Westfall, and a number 
of nutritional variants, the tools may now be at 
hand to learn of the mechanisms involved in these 
transformations, to attempt to control and induce 
them, and to test for the transfer of genetic ma- 
terials between cells. Recently Dr. Sanford and 
her associates have reported additional studies 
on the development in vitro of hereditary differ- 
ences in morphology and further alterations in 
arginase and Jeta-glucuronidase activities within 
a clone of mouse tumor cells. By means of clonal 
analysis additional hereditary alterations in mor- 
phology, level of enzyme activity, tumor-pro- 
ducing capacity, and chromosomal constitution 
have been determined among clonal derivatives. 
Last year an initial attempt to identify "hy- 
bridization" of cells produced by transfer of 
genetic materials between cells was not success- 
ful, but encouragement for new attempts now 
comes from work by Barski, Sorieul and Corne- 
fert, who, using clones obtained from Dr. San- 
ford, have demonstrated "hybridization" of cells 
as evidenced by chromosome markers (see also 
the report below in the section on Nucleic Acids 
by Drs. Law, Bradley, and Roosa) . 

Dr. Virginia J. Evans has studied three series 
of fresh C3H embryo explants cultured in me- 
dium NCTC 109 and 10 percent horse serum to 
determine the time and incidence of transforma- 
tion to malignancy as determined by intraocular 



NATIONAL CANCER INSTITUTE 



51 



injection following in vitro growth periods of 
different lengths. Tumors occurred after the 
cells were in vitro only 176 days, 147 days and 
138 days, respectively, for the three series. 
Growth acceleration of the cultures occurred some 
4 to 8 weeks prior to the time the transformation 
took place in vitro. The tumors produced in the 
eye were fibrosarcomas, with latent periods rang- 
ing from 17 to 125 days. Exploratory chromo- 
some counts indicated that there were many 
heteroploid cells in the populations. Such infor- 
mation pinpoints rather sharply the time at which 
the investigator must look for leads in under- 
standing the transformation and provides a fairly 
clear-cut calendar for other studies requiring 
knowledge regarding the state of the cells in 
culture. 

Similar studies with medium NCTC 109 with- 
out added serum have been done with implanta- 
tion intraocularly after 26 to 211 days in vitro. 
Eesorption of all implants has occurred to date. 
It should be pointed out that under these condi- 
tions* the rate of growth in vitro has been found 
to be slow, with death occurring and waves of 
new cells growing out. 

Fibroblasts obtained by implanting small pieces 
of epigastric fat pads in double millipore filter 
chambers (porosity HA) which were then placed 
in the peritoneal cavities of mice and grown 
through 12 successive chamber transplant genera- 
tions have good growth in vitro. Implants into 
the eye survive, but do not proliferate. 

Dr. Sanford also continues to investigate sev- 
eral variables of cells grown in culture beyond 
short term periods through study of effects of 
vitamins varied, singly or in combination, in their 
concentrations. Medium NCTC 109, a protein- 
free, chemically defined medium that supports 
long-term proliferation of cells grown in culture 
and Was developed a few years ago, is extremely 
Complex in composition. Although a simplified 
medium, NCTC 118, is an improvement, it never- 
theless contains 18 vitamins. As reported last 
year, when each of the 18 vitamins was individu- 
ally deleted from the medium, 5 vitamins (cal- 
cium pantothenate, choline chloride, niacinamide 
or niacin, thiamin and riboflavin) were identified 
as essential for cell survival. Vitamins not ap- 
parently required for cell survival, but which 
increased rates of cell proliferation were folic 
acid and pyridoxal (or pyrodoxine). 



During the past year, two new media were 
evaluated, and the interrelationship of vitamin 
B12, folic acid and thymidine were studied in 
their effects on cell proliferation and growth. 
Medium NCTC 119, containing only seven vita- 
mins, failed to support cell survival for more 
than 2 or 3 weeks. Additional studies with five 
other vitamins showed an importance of biotin 
which could not be demonstrated in the presence 
of all 18 vitamins. Investigations with medium 
NCTC 120, which contains biotin, showed that 
the cells, though proliferating as rapidly as the 
controls in medium NCTC 118, were fragile and 
could not withstand the stirring used to set up 
quantitative experiments. When vitamin Bi 2 , 
folic acid or thymidine were deleted individually 
from the medium, no detectable effect on cell sur- 
vival or proliferation was noted. "When all three 
were deleted, however, growth was poor, and en- 
larged cells with abnormal nuclei resulted. In 
the absence of folic acid and Bi 2 , some reduction 
in growth was noted. A more marked reduction 
in growth occurred in the absence of thymidine 
and folic acid. In the latter medium containing 
only B12, cells increased in nuclear mass and cell 
numbers decreased to a critical point. Although 
a few cells appeared to adapt to the medium, 
growth lasted only 4 to 6 weeks. 

One nutritional variant which does not require 
pyridoxine or pyridoxal was isolated, but the 
cells are exceedingly fragile. 

Immunological Aspects. Utilizing red cell ag- 
glutination techniques, Dr. M. A. Fink and Dr. 
Evans found that in guinea pigs antibodies were 
produced which were specifically reactive with 
only homologous erythrocytes when cultured mon- 
key or human cells carried in culture for long 
periods of time were administered. Surprisingly, 
however, neither fresh monkey nor fresh human 
cells incited the production of antibody reactive 
with homologous erythrocytes. Tests for delayed 
hypersensitivity revealed that both the tissue cul- 
tured and fresh tissues contained common anti- 
gens. In a comparison of fresh vs. cultured 
human epithelium, it was noted that pigs injected 
with fresh tissue reacted to human serum, while 
those injected with cultured tissue did not. In 
tests for immediate hypersensitivity (Schultz- 
Dale technique) there was again evident a marked 
diminution or absence of reactivity in the uteri 



52 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



from pigs injected with tissue cultured cells, but 
not in those from pigs injected with fresh tissue, 
when brought in contact with homologous serum. 
These changes in antigenic characteristics seen 
in cells grown in culture suggest the possibility 
of loss of surface antigen with an uncovering or 
increase in availability or potency of the antigen 
responsible for hemagglutinating antibodies. 
Should histocompatibility antigens be involved, 
special culture techniques might permit growth 
of cells containing viruses from one species and 
subsequent transfer to experimental animals of a 
different species — another approach of importance 
to the study of human tumors. The above find- 
ings also confirm the usefulness of the Brand 
hemagglutination technique in determining the 
species of origin of cultured cells, a matter of 
considerable importance in establishing and oper- 
ating tissue banks. 

Enztmologt. Dr. D. B. McN. Scott (University 
of Pennsylvania) and Dr. Sanford have evalu- 
ated certain oxidative enzymes involved in glucose 
metabolism in a cloned cell line that produces 
tumors rapidly upon implantation in mice and in 
three cloned cell lines that produce tumors slowly 
if at all upon implantation in mice. The former 
showed nearly three times the hexokinase activ- 
ity, four times the glucose-6-phosphate dehydro- 
genase activity, and five times the phosphoglu- 
conate dehydrogenase activity compared with the 
latter. 

Nine long-term mouse cell culture strains and 
two human ones were all found by Dr. Westfall 
to produce a^Aa-keto-glutaric and pyruvic acids, 
an indication that all of them posses active trans- 
aminating enzymes needed for the formation of 
these keto-acids, or, in the case of pyruvate, that 
it was formed from glucose. On the other hand, 
catalase activity in twelve long-term mammalian 
tissue cell strains, including three derived from 
mouse liver epithelial tissue, was found to be very 
low. These findings supply additional evidence 
to that previously obtained by Dr. Westfall which 
indicates no necessary parallelism among the vari- 
ous enzyme systems in cells grown for long pe- 
riods in culture. Some enzyme systems not 
necessary for cell maintenance disappear; others 
remain active and may be necessary for cell sur- 
vival growth. 

Tumors arising from injection into mice of 



some of the mouse cells also had low catalase 
activity. Hepatomas arising from injection of 
the hepatoma strain of mouse cells likewise had 
low activity. Furthermore, the liver catalase of 
the hosts bearing these hepatomas was not de- 
pressed. 

Studies on Proteins and Nucleic Acids 

The great importance of proteins and nucleic 
acids in the control of functions of normal and 
abnormal cells and of whole organisms is at- 
tested by the vast amount of work, in many cases 
by leading scientists, on the subject. Like other 
members of the staff of the National Institutes 
of Health, investigators in the National Cancer 
Institute are making significant contributions to 
the knowledge of these important materials. The 
fundamental studies on proteins and nucleic acids 
provide a base upon which further understanding 
of disease processes, including cancer, are built. 
Contributions to the separation and characteriza- 
tion of complex protein mixtures made by Drs. 
H. A. Sober and E. A. Peterson have had far- 
reaching influence on research conducted on the 
proteins and nucleic acids. 

Drs. Peterson and Sober have prepared two 
new cellulose ion exchangers (designated ECA- 
cellulose and ECTHAM-cellulose, respectively) 
for use in the chromatography of substances that 
are too tightly bound to the previously available 
adsorbents. Both possess basic groups that are 
weaker than those of DEAE-cellulose or ECTE- 
OLA-cellulose. They have been used successfully 
in the chromatography of ribosomes and soluble 
ribonucleoprotein. New elution gradients and 
new buffer species have been evaluated. Of spe- 
cial interest is tris-succinate buffer since it gives 
much sharper peaks than phosphate buffer in the 
elution of serum albumin and, in studies on trace 
metals, is completely combustible in the carbon 
arc used for metal analyses. 

Sephadex is becoming increasingly useful as a 
dialysis replacement; furthermore, the combina- 
tion of Sephadex with the cellulose ion exchangers 
seems like a profitable avenue of approach since 
a multitude of stages of continuous salt displace- 
ment can be obtained by such a mixture. 
Protein Characterization. Drs. Peterson and 
Sober, utilizing gradient and stepwise elution 
techniques in chromatographic fractionation on 



NATIONAL CANCER INSTITUTE 



53 



cellulose ion exchangers, have determined addi- 
tional properties of serum fractions. 

Chromatography of crystalline bovine plasma 
albumin (BPA) on DEAE-cellulose with a suit- 
able gradient yields a skewed peak, suggesting 
heterogeneity. Treatment of the BPA with cetyl- 
trimethylammonium bromide (CTAB) before 
chromatography preferentially converts the ear- 
lier, major portion of the peak into complexes 
having little or no affinity for the adsorbent. As 
the major component is thus diminished, the re- 
sidual, presumably unaltered, albumin can be 
seen to comprise at least 4 components. Unlike 
the CTAB complex of human serum mercaptal- 
bumin, that formed with BPA is largely dis- 
associated by dialysis. 

Chromatography of an old sample of lyophil- 
ized Red Cross human Fraction V gave two ap- 
proximately equal peaks. The first was pure 
monomer; the second was 70 percent dimer and 
trimer. Fresh Eed Cross Fraction V, on the 
other hand, appeared as a single, highly skewed 
peak containing little or no dimer. 

Esterase activity of highly purified albumin, 
which was first demonstrated by Kramer, may 
represent a true metabolic function of the albu- 
min, or it may be a fortuitous consequence of the 
structure of the molecule. All of the chromato- 
graphic fractions of BPA showed equal esterase 
activity, indicating that the activity is not at- 
tributable to an enzyme impurity. The addition 
of CTAB caused a three-fold increase in esterase 
activity, although the same concentration of this 
compound had no effect on the substrate in the 
absence of albumin. 

Progress on the isolation and characterization 
of the metal proteins of serum has remained slow 
because of difficulties in insuring the absence of 
contaminating metals in the solutions and equip- 
ment used. Use of plastic materials and the 
preparation of metal-free reagents should allow 
greater progress during the coming year. 

Fractionation of the antihemophilic factor 
(Factor VIII, AHF) is progressing very satis- 
factorily through collaboration with Drs. Mar- 
tinez-Canaveri and Casillas of Argentina. AHF 
has been purified with excellent recovery and the 
ensuing lability of the activity has been avoided 
in part by the addition of glucose. Although 
still in the preliminary stage, it also seems pos- 
sible to adsorb the AHF directly on DEAE- 



cellulose from undialyzed and undiluted plasma 
from which interfering materials have been re- 
moved by prior aluminum hydroxide adsorption. 
This simple procedure shows considerable prom- 
ise of easily providing AHF preparations that 
would be suitable for clinical use. Cooperative 
studies with the Protein Foundation are being 
instituted. 

Dr. R. TV. Hartley, Jr., working with Drs. 
Sober and Peterson, has studied crystalline bovine 
plasma albumin and a number of its derivatives 
by chromatographic fractionation. In the high 
degree of resolution obtained, the most striking 
result has been the separation of mercaptalbumin 
and nonmercaptalbumin. Subsequent experiments 
indicated that nonmercaptalbumin, after reduc- 
tion with mercaptoethanol, behaved chromato- 
graphically as mercaptalbumin. This would 
indicate that some of the chromatographic differ- 
ences observed are related to the presence or 
absence of a free SH group. Studies are under 
way to determine the position of this group in 
the albumin molecule. Albumin dimers have 
been found to be heterogeneous chromatographi- 
cally as well as with respect to reduction by 
mercaptoethanol to the monomer. Dimers can 
be produced from the albumin monomer even in 
the absence of free SH groups and appear to be 
S-S dimers in that they are reducible by mer- 
captoethanol. Such studies are of importance in 
learning more of the physical and chemical forces 
involved in maintaining the native configuration 
and activity of many proteins. 

Protein Synthesis Studies. Dr. E. L. Kuff con- 
tinues his investigation of the cytochemical or- 
ganization of normal and malignant cells, with 
particular reference to the problem of protein 
synthesis and replication of intracellular compo- 
nents. He has used as a particularly valuable 
source of cells the plasma cell tumors, which 
have been shown by Drs. M. Potter and J. Fahey 
to produce and secrete into the circulating blood, 
large quantities of myeloma proteins while simul- 
taneously maintaining a high rate of mitosis. 
Electron microscopy by Dr. A. J. Dalton has 
revealed the presence of an extensive endoplasmic 
reticulum and highly developed Golgi systems 
within the cells. Ribonucleoprotein (RNP) par- 
ticles were extremely numerous, both in associa- 
tion with the neoplasmic reticulum and as appar- 



54 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ently free entities in the cell sap. Fractionation 
of plasma cell tumor homogenates by standard 
methods of differential centrifugation gave two 
subfractions of the entire microsome fraction: 
one made up primarily of free RNP particles, 
and the other composed of fragments of the endo- 
plasmic reticulum (membranes) with associated 
RNP particles (vide supra the collaborative work 
with Dr. M. Potter on the antigenic properties 
of these fractions). 

Viruslike particles, regularly seen in neoplastic 
mouse plasma cells localized exclusively within 
the endoplasmic reticulum, have been reported 
by Dr. Dalton. Through the use of the nonionic 
detergent, TritonxlOO, it has been possible for 
Dr. Kuff to disrupt the membrane component of 
the isolated tumor microsomes in such a way as 
to liberate the virus-like particles in morphologi- 
cally unaltered form. Evaluation of the biologi- 
cal activity of these preparations is under way 
with Drs. Dalton and Law. 

Through the use of a density gradient frac- 
tionation method, Dr. Kuff has been able to iso- 
late Golgi membranes from a mouse plasma cell 
tumor. With Dr. R. F. Ziegel, it has been shown 
that the tumor Golgi fractions resemble those 
previously obtained from epididymus, both in 
their morphology and in their high content of 
lipids. The rate of incorporation of P 82 into the 
phospholipids of isolated Golgi fractions of epi- 
didymus has also been compared with the corre- 
sponding rates for other intracellular fractions. 
For the relatively long incorporation periods thus 
far studied (1 to 3 hours), there was little dif- 
ference between the Golgi and other cytoplasmic 
fractions. 

These studies, particularly the immunochemical 
studies of plasma cell tumors with Dr. Potter 
mentioned above, should prove to be very valuable 
in investigating the relationships between the 
synthesis of specific secretory proteins and that 
of the actual cellular proteins themselves. Espe- 
cially important will be the studies on the micro- 
somal RNP particles, since they may represent 
the sites of synthesis of these proteins. If arti- 
ficial redistribution of protein during the prepara- 
tion of fractions can be ruled out or eliminated, 
the question can then be investigated whether the 
myeloma globulins are associated with all of the 
tumor RNP particles or only with those attached 
to the endoplasmic reticulum. 



Drs. Peterson and Kuff have adsorbed rat liver 
microsomes isolated by differential centrifugation 
on a cellulose ion exchanger, allowing glycogen 
particles and residual protein to pass through 
unadsorbed. Because microsomes exhibit an un- 
usual tendency to establish additional bonds with 
the adsorber, difficulties were encountered in the 
chromatography of these cytoplasmic fragments. 
However, with modification of techniques, clean 
preparations of particles having a sedimentation 
constant of 77S were obtained. Soluble ribonu- 
cleoprotein can also be obtained by chromatog- 
raphy of a clear, particle-free supernatant rat 
liver homogenate. Phenol extraction of the ribo- 
nucleoprotein yielded protein and an "S-RNA." 
The latter, upon re-chromatography, behaved 
identically with the original ribonucleoprotein. 
Further studies are under way to elucidate the 
reasons for the identical chromatographic be- 
havior and to determine the completeness of the 
removal of protein. These techniques promise 
to permit separation of these important cell par- 
ticulates under milder conditions than any yet 
reported. 

Dr. M. Rabinovitz, as part of continuing at- 
tempts to introduce in vitro "biochemical lesions" 
within the protein synthetic pathway in normal 
and tumor tissues, has synthesized two antime- 
tabolites, 4-thiamethione (antimetabolite of meth- 
ionine) and S-carbamylcysteine (antimetabolite 
of glutamine), and has studied their inhibition 
of the incorporation of labeled amino acids into 
protein. The former inhibits incorporation of 
methionine, leucine, valine, lysine, and phenylala- 
nine, and methionine does not prevent the inhi- 
bition. Glucose both prevented and reversed the 
inhibition, suggesting an interference with the 
oxidative energy supply supporting protein syn- 
thesis. The latter inhibited the incorporation of 
glutamine and other amino acids into protein. 
The inhibition of glutamine incorporation, which 
was not reversed by glutamine, was greater per- 
centage-wise in the presence of higher concentra- 
tions of glutamine, and this inhibition was shown 
to be due to interference with the general stimu- 
lation of amino acid incorporation known to be 
produced by glutamine. Dr. Rabinovitz has also 
found that the antibiotic, puromycin, can inhibit 
the incorporation of amino acids into ribosomal 
protein, but not into soluble protein of cells. 



NATIONAL CANCER INSTITUTE 



55 



Amino Acid and Peptide Synthesis. The prep- 
aration of a series of alpha-ammo-beta, hydroxy 
acids (in a single step reaction which involved 
the treatment of an alkaline solution of an amino 
acid with an aldehyde, in the presence of catalytic 
amounts of cupric ion) was reported last year by 
Drs. T. T. Otani, N. Izumiya, S. M. Birnbaum, 
and M. Winitz. Additional hydroxyamino acids 
have now been synthesized, as well as some acyl 
derivatives, and resolution of alpha-methyl-DLi- 
serine and aZpAa-ethyl-DL-serine were attempted. 
Unexpectedly, the N-chloroacetyl derivative of 
the former compound could be resolved only par- 
tially by the usual enzymatic procedure since the 
hydrolysis continued on beyond the customary 
50 percent point to reach 100 percent hydrolysis, 
indicating that the enzyme could not discriminate 
between the asymetry of both optical isomers of 
the molecule (i.e., the hydroxymethyl and methyl 
groups were not sufficiently dissimilar) . Partial 
resolution could be demonstrated, however, if the 
reaction was stopped slightly beyond 50 percent 
hydrolysis. The resolution of aZpAa-ethyl-serine 
was accomplished in the usual manner. 

Growth inhibition studies with E. coli were 
undertaken by Dr. Otani with several of the syn- 
thesized compounds. Inhibition was observed to 
be most effective with Jeta-hydroxy-DL-aspartic 
acid, &eta-hydroxy-DL-leucine, and L-O-methyl- 
threonine. 

Dr. Winitz has successfully synthesized and 
isolated the epimeric mixture of <feZfa-hydroxy- 
lysine in about 50 percent over-all yield. Separa- 
tion of the diastereomers was achieved by 
fractional crystallization of their alpha-cholovo- 
acetyl-epseZon-carbobenzoxy derivatives. Resolu- 
tion was achieved enzymatically with cobalt- 
activated acylase I. 

Drs. N. Izumiya (Visiting Scientist) and 
Winitz have prepared the four steroisomers of 
valyllysine anhydride and of phenylalanyllysine 
anhydride to obtain additional information on 
the comparison of chemical structure and biologi- 
cal activity in a group of compounds represented 
by the polypeptide antibiotics, gramicidin S and 
tyrocidin A. These antibiotics have the same 
features in common: a) a basic character due to 
the presence of one or more diamino acid resi- 
dues; b) a cyclic conformation; and c) at least 
one amino acid residue of the D-configuration. 
Although the synthesized compounds also possess 



these characteristics, no antibiotic activity was 
found against either Gram-positive or Gram- 
negative bacteria. Moreover, they were not sus- 
ceptible to the hydrolytic action of trypsin or 
chymotrypsin. 

Nucleic Acids. Drs. Sober and G. Rushizky 
(Visiting Scientist) are exploring modifications 
of chromatographic techniques that will permit 
separation of fractions containing large nucleo- 
tide sequences resulting from enzymatic digestion 
of nucleic acids. All of the mono-, di-, and tri- 
nucleotides resulting from ribonuclease digests of 
ribonucleic acids have previously been separated. 
Fractionation studies in collaboration with Drs. 
L. Heppel and M. Singer (NIAMD) of adenylic 
acid polymers from the hexa- to the deca-nucleo- 
tide and larger fragments, coupled with analyti- 
cal paper chromatography, have indicated the 
presence of contaminating enzyme activities in 
the original enzyme preparation used to degrade 
the polyadenylic acid polymer. Homologous 
oligonucleotides are being used as model sub- 
stances for nucleotide separations. Such studies 
are of considerable importance in the investiga- 
tion of nucleic acids in that they serve to char- 
acterize the enzymatic action, to characterize the 
nucleic acid itself in terms of its nucleotide dis- 
tribution and sequence, and to obtain a variety 
of pure oligonucleotides which can subsequently 
be used in physical-chemical and enzymatic 
studies. 

Drs. Rushizky and Sober have prepared pan- 
creatic ribonuclease digest of a high molecular 
weight ribonucleic acid from yeast and from 
tobacco mosiac virus (supplied by Dr. C. A. 
Knight, Virus Laboratory University of Cali- 
fornia, Berkeley). The digests were fraction- 
ated by a two-dimensional mapping procedure 
which combines paper electrophoresis and paper 
chromatography, and the fractions so isolated 
were further examined by physical-chemical and 
enzymatic methods. Comparisons were made 
with the complete digests. The following gener- 
alizations were obtained: a) oligonucleotides are 
released before the pyrimidine mononucleotides; 
b) with the pyrimidine mononucleotides, cytidylic 
acid was released before uridylic acid; and c) in 
each case, the cyclic intermediate of cytidylic acid 
or its oligonucleotides were converted to the ter- 
minal 3'-form before the corresponding uridylic 



56 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



acid compounds. Pancreatic ribonuclease has 
been found to have a preference for the enzymatic 
hydrolysis of pyrimidine linkages adjacent to 
purine linkages, i.e., these linkages are split be- 
fore pyrimidine-pyrimidine bonds. This prefer- 
ential specificity may be of considerable assistance 
in providing large and homogenous oligonucleo- 
tides in pure form which will aid in the determi- 
nation of nucleotide sequences in nucleic acids. 

As a further aid to work aimed at determining 
the structure of nucleic acids, Drs. Rushizky and 
Sober have purified two nucleases, "adenylase" 
and "guanylase," by means of conventional pro- 
tein fractionation techniques, including precipi- 
tation and ion exchange chromatography. Both 
enzymes have been purified some 400-fold, and 
each is free of the other activity when tested at 
an enzyme-substrate ratio of 1 :100. Since these 
two enzymes appear to hydrolyze adenylic and 
guanylic acid nucleotide linkages respectively, 
they will, especially when purified further, be 
particularly useful as tools for the specific cleav- 
age of nucleotide significances of ribonucleic acid 
and provide therefore a complementary technique 
to that provided by the pyrimidine specificity of 
pancreatic ribonuclease. 

Dr. Mary Maver, with Miss Antoinette Greco, 
has been purifying nuclease preparations derived 
from normal and neoplastic tissue, and has fur- 
ther characterized their activities. Utilizing beef 
spleen and liver, rat liver, two transplantable rat 
hepatomas and a rat lymphosarcoma, she has 
obtained ribonuclease preparations by means of 
chromatography whose activities are character- 
ized by: (a) their pH optima on highly polymer- 
ized rat liver ENA; (b) the products isolated 
from their hydrolysates when cyclic purine and 
pyrimidine nucleotides are used as substrates; 
(c) the products of hydrolysis isolated in work 
with Dr. Rushizky from hydrolysates of yeast 
RNA by paper electrophoresis followed by paper 
chromatography; (d) the products of their hy- 
drolyses of polyadenylic and polycytidylic acids, 
and (e) the effects of Mg ions and heat upon 
their RNAase activities. Two enzymatic activi- 
ties which fractionate closely with the RNAase 
activities have been removed by repeated fraction- 
ation on carboxymethyl cellulose columns: (a) 
an acid phosphatase which preferentially dephos- 
phorylates the 3'-adenylic acid and, (b) a phos- 
phodiesterase, without RNAase activity, which 



hydrolyzes cyclic adenylic acid to yield the 
2'-adenylic acid. These enzymes are much more 
active in calf spleen and liver and rat hepatoma 
3683 than in normal rat liver. 

An active "acid" RNAase with a pH optimum 
of 5.7 has been isolated which does not hydrolyze 
cyclic adenylic acid, but does hydrolyze cyclic 
cytidylic acid and polyadenylic and polycytidylic 
acids to yield the 3'-derivatives. With yeast or 
rat liver RNA as a substrate, both purine and 
pyrimidine linkages are broken and no core re- 
mains. The alkaline RNAase of spleen hydro- 
lyzes cyclic adenylic and cytidylic acids to give 
the 3'-acids. Rat liver has been found to have a 
RNAase with a pH optimum of 6.5-6.7 besides 
the activities with optima of pH 5.7 and 7.3-7.8. 
The 6.5-6.7 RNAase is masked in crude prepara- 
tions by an inhibitor which can be removed by 
treatment of the preparations with heat or para^ 
chloromercuribenzoate. The RNAases of liver 
are considerably less active than those of other 
tissues studied. 

Dr. Maver and Miss Greco have also been 
studying the sulfhydryl dependency of the RNA- 
ase activities of liver. As has been shown by 
others, para-chloromercuribenzoate (CMB) acti- 
vates the alkaline RNAases by combining with 
an inhibitor. When this inhibitor has been re- 
moved by the chemical purification procedures 
used to obtain the more active nuclease prepara- 
tions, often the alkaline as well as the acid 
RNAase activities can be activated (about 20 per- 
cent) by cysteine and inhibited by CMB. The 
acid RNAase is much more sensitive to CMB 
than the alkaline RNAases. The inhibitions of 
RNAase activities by CMB can be reversed by 
excess cysteine when the right quantitative con- 
ditions prevail. Spectrophotometric assays done 
according to Boyer show that the combination of 
the SH of the RNAase protein with the CMB 
is directly correlated in timed reactions with the 
inactivation of the enzyme. These studies also 
should not only aid in elucidating the nucleotide 
sequence of nucleic acids, but the sulfhydryl sen- 
sitivity noted above may have important impli- 
cations in the regulation of RNAase activity 
which may control RNA production for protein 
synthesis and in the liberation of energy-yielding 
or accepting nucleotides. 

Extensive and detailed studies on deoxyribonu- 
cleic acid (DNA) and neutral deoxy ribonuclease 






NATIONAL CANCER INSTITUTE 



57 



(DNAase I) have been made by Dr. J. Shack. 
Various conditions that affect enzyme activity 
(pH, substrate concentration, type and concen- 
tration of cations and of nonspecific salts) are not 
independent but rather are interdependent vari- 
ables. Contrary to many earlier reports in the 
literature, it was found that proper control of 
these variables yields a substrate concentration- 
activity relation which corresponds to classical 
Michaelis-Menten kinetics. It was also found 
that the rate of hydrolysis of DNA falls off rap- 
idly to a very low value when less than half of 
the susceptible bonds are broken. It appears that 
DNA contains linkages of widely different sus- 
ceptibility to the enzyme and that the rate falls 
off as the more susceptible linkages are broken, 
rather than because of reduction of substrate con- 
centration or inhibition by products of the reac- 
tion. Comparison of results with native and 
denatured DNA indicates that differences in sus- 
ceptibility are not dependent on the specific 
secondary structure possessed by native but not 
by denatured DNA, but are more probably related 
to the sequence of nucleotides in the vicinity of 
the particular bond. 

Dr. E. Kielley, utilizing the assay method of 
Dr. Kornberg and associates, has characterized 
some of the conditions influencing deoxynucleo- 
tide kinase activities in liver homogenates of mice 
and rats and has studied the extent to which 
these enzymes can catalyze the synthesis of de- 
oxynucleoside di- and tri-phosphates (apparently 
the direct precursor substances of DNA) in nor- 
mal mature cells. From studies of substrate 
concentration on other deoxynucleotide kinase 
activities, one can conclude that the enzyme ac- 
tivity levels of deoxycytidylic acid, deoxyguanylic 
acid, and deoxyadenylic acid kinases are com- 
paratively high and would not therefore be limit- 
ing factors in DNA synthesis. The activity of 
thymidylic acid kinase is somewhat low and addi- 
tional work is required to determine if it can be 
considered a limiting factor in DNA synthesis. 
Preliminary results with tumor tissues indicate 
that the course of deoxynucleotide tri-phosphate 
synthesis in tumors is radically different in some 
respects from that found in normal liver. 

Deoxycytidine diphosphocholine (d-CDP-C) 
and deoxycytidine diphosphoethanolamine (d- 
CDP-E) were isolated from the Novikoff hepa- 
toma by Dr. W. C. Schneider 2 years ago. Assay 



conditions developed and isotopically labeled sub- 
strates synthesized have permitted initiation of 
studies of the factors responsible for the concen- 
trations of these compounds which are higher in 
hepatoma and regenerating liver than in normal 
liver. The enzymatic activities related to the 
synthesis of d-CDP-C are considerably higher in 
homogenates of Novikoff hepatoma and of re- 
generating liver than in those of normal liver. 
These compounds may prove to be of importance 
in DNA synthesis since they may lie on the path- 
way for conversion of ribonucleotides to deoxy- 
ribonucleotides. 

With Drs. E. T. Bradley (last year an Anna 
Fuller Fund Fellow) and E. Eoosa (now at the 
Wistar Institute), Dr. L. Law has provocative 
preliminary evidence obtained in three separate 
instances indicating that stable, DNA-induced 
transformations in mammalian cells may be de- 
monstrable. P-88 sensitive cells incubated in 
vitro with DNA prepared from cells with a high 
level of 8-azaguanine resistance developed resist- 
ant colonies at a frequency 20 to 40 times that 
observed in sensitive control cultures. 

Enzymatic Studies 

Dr. S. M. Birnbaum and Mr. M. C. Otey have 
made comparisons of the rate of disappearance of 
various D-amino acids in rats (interpreted as 
in vivo oxidation) and the rate of oxidation in 
vitro of corresponding D-amino acids. Small 
amounts of the compounds were force fed to 
young rats in 6 divided doses over a 2-day period. 
The excreta were quantitatively collected and 
were assayed for the D-amino acid. The total 
excretion subtracted from the amount fed was 
assumed to give a measure of the in vivo oxida- 
tion. Although the fraction "oxidized" varied 
from 40 to almost 100 percent, depending on the 
nature of the amino acid administered, this per- 
centage value was nearly constant for a given 
D-amino acid at whatever level it was fed. Study 
of the specific and total oxidative activity of rat 
kidney and liver homogenates against a series of 
D-amino acids revealed : a) the relative specificity 
of the two tissues is similar if not identical in 
this respect, and b) there is no apparent relation 
between the in vitro and in vivo oxidative rates 
for D-amino acids. This latter observation raises 
some question as to the physiological interpreta- 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tion of the significance of in vitro enzyme meas- 
urements. 

In previous studies on the isolation of cell 
particulates by Dr. R. E. Greenfield, Jr., it was 
noted that the addition of the high molecular 
"weight polymer, polyvinylpyrrolidone (PVP), to 
solutions of sucrose resulted in a marked protec- 
tion of cellular particulates. He has now made 
use of PVP-sucrose solutions and an interesting 
use of proteolytic enzymes administered intra- 
venously to obtain suspensions of individual cells 
from different tissues. As suspension of single 
cells was obtained which contained 15-25 percent 
of the total liver nitrogen when the in vivo injec- 
tion of trypsin and a fractional centrifugation 
procedure were used. This fraction was relatively 
free of cell debris and was made up predominantly 
of parenchymal cells. The staining properties of 
the cells would indicate that a majority of them 
are alive : i.e., over 90 percent of the cells in most 
suspensions did not take up trypan blue or nigro- 
sin and nearly all of the cells took up tetrazolium 
under anerobic conditions. The ease with which 
single cell fractions could be isolated from other 
organs varied markedly. Cells could be isolated 
in high yield from spleen, thymus, lymphosar- 
coma, and hepatoma. Only after multiple trypsin 
injections could the kidney be separated into cells, 
and even then the yield of cells was smaller than 
that found for the preceding organs. Following 
a single injection of trypsin the kidney separated 
into glomeruli and various lengths of tubules and 
blood vessels. Although muscle fibers which had 
been subjected to the in vivo trypsin injection 
could be easily teased apart, they had to be cut 
from the tendons. The brain and fibrous tumors 
separated into cells rather poorly, and the heart 
tissue did not separate at all. 

Little change other than petechiae in the lungs 
could be seen on microscope examination of the 
organs following a single in vivo injection of 
trypsin. The animals did not appear in pain and 
remained healthy if permitted to survive. 

When liver cell suspensions were kept at 0° in 
20 percent PVP-10 percent sucrose solutions, the 
activity of aldolase, lactic acid dehydrogenase and 
catalase over an 8-hour period was nearly the 
same on a nitrogen basis as that found in the liver 
of origin. When the isolated liver cells were sus- 
pended in culture media at 38°, certain of these 
enzymes were lost. When 20 percent PVP-10 



percent sucrose was added to NCTC 109, a pro- 
tein-free, chemically defined medium, 80 percent 
of the lactic acid dehydrogenase was lost from 
the cells to the media in one hour while over 
90 percent of the aldolase and the catalase could 
be recovered from the cells. Aldolase was rapidly 
lost when sucrose was removed from the media. 
The addition of methocel or serum did not pre- 
vent the rapid loss of enzymes. 

Drs. M. Chirigos and A. Goldin have studied 
a transaminase system, which mediates the trans- 
fer of alpha-&mino groups of several alp?ia-&miao 
acids to jcwa-hydroxyphenylpyruvate with the 
formation of tyrosine, in liver and in spontaneous 
and transplanted tumors. With the amino acids 
employed, a low degree of similarity in trans- 
aminating pattern was observed among the tu- 
mors when compared with the pattern for liver. 
Tumor extracts showed greater transaminating 
activity with glutamic acid than with glutamine, 
in contrast to liver preparations which exhibited 
similar activities with both compounds. The 
study also indicates enzyme systems not previ- 
ously reported exist both in liver and in the 
tumors. 

Dr. Helen Dyer has been studying glutamic- 
oxalacetic transaminase (GOT) activities of sev- 
eral tumors, including the Morris 5123 hepatoma, 
in a number of strains of rats and the activity 
in their sera. The level of serum GOT activity 
was not reduced during the first two months of 
feeding of several hepatocarcinogenic aromatic 
amines nor the chemically related nonhepatocar- 
cinogenic compounds in the dosage used for the 
induction of tumors. GOT activity of livers of 
animals bearing tumor transplants did not differ 
significantly from those of normal animals. Al- 
though several transplanted tumors showed a 
lower GOT activity than normal liver, the Mor- 
ris hepatoma 5123 had a higher activity. Fur- 
thermore, the GOT activity of sera of rats bear- 
ing hepatoma 5123 was considerably above that 
of normal rats, whereas the sera of rats bearing 
several other transplanted tumors was within the 
normal range. As the hepatoma 5123 grew in 
size, the serum transaminase activity increased 
accordingly. The serum level can also be raised 
by the intraperitoneal injection of the supernatent 
layer of a centrifuged homogenate of hepatoma 
5123. Very little GOT activity was detected in 
the urine. The results suggest that the increased 



NATIONAL CANCER INSTITUTE 



59 



serum GOT activity originates in the tumor tis- 
sue and that it accumulates in the blood because 
it can be excreted only slowly by the kidneys. 

An intriguing experiment has been performed 
by Dr. D. Burk and Mr. J. Hunter in which 
thermophilic chlorella cells were introduced into 
the peritoneal cavity of mice. Severe and chronic 
hypoglycemia occurred that resulted in the death 
of the animal. Cells washed from the peritoneal 
cavity of the mouse, where they had remained 
for 17 hours, grew vigorously when placed in 
proper medium, atmosphere and light. The hypo- 
glycemia could be corrected by administration of 
glucose. This experiment followed studies on the 
inhibition of anaerobic and aerobic glycolysis of 
Ehrlich Krebs-2 mouse ascites cells by the pres- 
ence of thermophilic chlorella cells grown simul- 
taneously with the tumor cells in Krebs-Einger 
medium. Because the algae consumed glucose at 
a rate several times faster than the cancer cells, 
the inhibition appears to result from low avail- 
ability of glucose for the cancer cells. The algae, 
like the tumor cells, can produce lactic acid from 
glucose, but D-lactic acid instead of L-lactic acid 
is formed. 

Dr. H. Kahler, with Mrs. B. Moore, extended 
his earlier studies on the pH of tumor tissue. 
Following administration of glucose to rats, the 
pH fell rapidly and returned to normal in tumor 
tissue at sites in the tumor with good blood cir- 
culation. An estimate of the blood circulation 
was made by parenteral administration of Lissa- 
mine green dye; tumor tissue stains green if the 
tumor has a good blood supply, remains white if 
the blood circulation is poor, and remains red if 
there are pockets of blood in a static condition. 

Drs. R. E. Madden and D. Burk have developed 
a procedure for comparing the metabolic be- 
havior of tumors in the solid and ascites forms 
that may facilitate metabolic and chemotherapeu- 
tic studies of solid human tumors and may aid 
in establishing single-cell-cloned tissue cultures 
of a variety of normal and malignant cells from 
human and animal tumors. Single-cell prepara- 
tions from several solid human and mouse tumors 
were prepared by stirring scissored tumor sections 
in media containing trypsin and deoxyribonu- 
clease, centrifuging the strained supernatant frac- 
tion and taking the sedimented cells up in medium 
not containing the enzymes. With the animal 
tumors, comparisons were made with single-cell 



preparations from solid tumors and from the 
same tumor grown in the ascites form in terms 
of metabolic activity and viability (quantitative 
in vivo mouse inoculation studies). The behav- 
iors of the two forms were essentially the same. 

Chemotherapy Investigations 

The search for chemical agents useful in the 
management of clinical cancer is under way on 
an unprecedented scale, and the stakes are high 
enough to warrant the tremendous efforts and 
large amounts of resources being expended. Two 
main approaches, not unrelated, have been em- 
ployed, and, despite arguments to the contrary 
by proponents of one approach or the other, both 
need to be vigorously advanced. One, for the 
most part an empirical approach, involves screen- 
ing of materials for their antitumor effects, the 
ultimate criterion being usefulness in the clinic. 
As yet, no satisfactory animal system screen has 
been developed that will predict the usefulness 
of drugs in the clinic, although some correla- 
tions exist between the responses of acute leu- 
kemia in children and leukemia L1210 in the 
mouse. Until much more data are available from 
studies on antitumor effects of various agents in 
both man and animal systems, it will be neces- 
sary to continue quantitative screening studies 
and to seek additional correlations. Investiga- 
tion of many parameters must be continued, such 
as different agents of various classes, different 
tumor types (with more emphasis on nontrans- 
planted tumors in animals), dosage schedules, 
routes of administration, combination therapy, 
immunological effects, and modifications of the 
host. The second approach, perhaps one in which 
the results might be of more far-reaching sig- 
nificance, but also perhaps slower in coming, is 
one aimed at understanding basic mechanisms of 
tumors, host-tumor relationships, and the mecha- 
nisms of action of chemical agents, both intrin- 
sic and extrinsic, on the tumor and the host. Ob- 
viously, knowledge gained in one approach will 
often be helpful to the other, but until a satis- 
factory screen is attained, the second approach 
needs heavy support, perhaps comparatively more 
than it has received in the large-scale chemo- 
therapy programs currently under way. 

The Biochemical Pharmacology Section, under 
the leadership of Dr. A. Goldin. conducts exten- 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sive programs in both approaches of cancer 
chemotherapy, in the former through effective use 
of the contract mechanism for a drug develop- 
ment and evaluation program. In this latter, 
which is under the direction of Dr. C. G. Zubrod, 
Clinical Director, NCI, and under the supervision 
of Dr. Goldin and Messrs. J. M. Venditti and 
G. A. Brandner, particular attention has been 
given to two factors that have limited the success 
of cancer chemotherapy: (a) the toxicity of 
drugs for the host; and (b) the capacity of the 
tumor to develop resistance to therapy. 

Relative to the former problem, the drug de- 
velopment and evaluation program is devoted 
primarily to determining quantitatively the rela- 
tive antitumor specificity of drugs of potential 
clinical interest. In particular, the program em- 
phasizes structure-activity relationships among 
compounds which emerge as "active" from pri- 
mary screens. Prolongation of the life-time of 
tumor-bearing animals is employed as the chief 
criterion for determining the relative therapeu- 
tic efficacy of drugs. Tumor size and body weight 
changes are, nevertheless, measured routinely. 
The program also investigates "active" com- 
pounds with respect to the optimal conditions for 
antitumor activity including the optimal sched- 
ule of treatment, route of administration, effect of 
combination therapy, etc. Such pharmacologic 
studies are needed to provide an accurate ap- 
praisal of the potential usefulness of a compound 
of interest. 

Studies of the problem of the development of 
resistance to treatment have been facilitated by 
the emergence of active compounds of various 
chemical classes which have the capacity to in- 
hibit growth at different biochemical sites and 
by the development of drug resistant sublines of 
tumors. The inclusion of studies of the effects of 
new compounds on drug resistant tumors is valu- 
able from two standpoints. Such studies can un- 
cover active compounds which may have clinical 
usefulness in cases which have become refractory 
to conventional treatment. In addition, drug re- 
sistant tumors are being utilized to explore pos- 
sible mechanisms of drug action. 

Tumor Assay Systems 

In the past, three tumor assay systems have 
been studied most extensively: 



The Advanced Leukemia L1210 Assay System. 
To date, 213 compounds (88 of which were tested 
in 1960) have now been evaluated for their ability 
to prolong the life of mice with systemic L1210. 
The most active compounds against this tumor 
have been the halogenated derivatives of ame- 
thopterin. For example, 3',5'-dichloroamethop- 
terin is about four times as active as amethop- 
terin. Extensive structure-activity relationship 
studies among folic acid derivatives have shown 
that appreciable antileukemic activity is limited 
to the 4-amino derivatives of this metabolite. 
However, substitutions in various other positions 
(e.g. alkyl substitutions in the 10-position or 
halogenation in the benzene ring) can profoundly 
alter the degree of antileukemic effectiveness 
among the 4-amino derivatives. Cytoxan, a cyclo- 
phosphoramide nitrogen mustard, displayed ac- 
tivity equal to that of amethopterin. Uracil 
mustard and a number of ethyleneimines showed 
moderate antileukemic activity, but were only 
about 50 percent as effective as Cytoxan. Of 35 
purines tested against advanced L1210, none ap- 
peared to be more effective than 6-mercapto- 
purine, which was 50 percent as active as ame- 
thopterin. Among the pyrimidines, 5-fluorouracil 
and its riboside displayed about equal effective- 
ness (about 40 percent of the amethopterin effect). 
4-Amino-pyrazolo (3,4-d) pyrimidine was about 
25 percent as effective as amethopterin. None of 
the various derivatives of this compound showed 
greater activity. Good activity was seen among 
the methylglyoxal-fo's-guanylhydrazones. Three 
such compounds tested displayed activities of 
about 65 percent. Glyoxal-6w-guanylhydrazone, 
itself, was relatively inactive. Among the anti- 
biotics, E-73 acetate, Streptovitacin A, and Ac- 
tinomycin D were the most active, being about 
30 percent as effective as amethopterin. 

Special studies with the advanced L1210 assay 
system have been directed toward investigations 
of factors which may alter the host-tumor-drug 
relationship, including the influence of the route 
of administration, schedule of treatment, effect 
of therapy with drug combinations, etc. It has 
been shown, for example, that the efficacy of 
therapy with 3',5'-dichloroamethopterin or 3'- 
bromo-5'-chloroamethopterin is substantially re- 
duced when the drug is given orally. Conse- 
quently, the superiority of the halogenated 
derivatives over amethopterin, seen on subcuta- 



NATIONAL CANCER INSTITUTE 



61 



neous administration, disappears when the drugs 
are given via the oral route. On the other hand, 
oral administration did not seriously reduce the 
effectiveness of 6-mercaptopurine. The methyl- 
g1yoxal-5is-guanylhydrazones were only slightly 
less effective orally than subcutaneously. Studies 
of the influence of the treatment schedule on anti- 
leukemic activity showed that Cytoxan was more 
effective when given weekly than when given 
daily, twice daily, every 4 days, or every 11 days. 
In general, the activities of the antibiotics tested 
were not appreciably influenced by the treatment 
schedule. The methylglyoxal-&is-guanylhydra- 
zones were more effective when given daily than 
when given every 4 days or as a single treatment. 
Combination therapy with amethopterin and 
Cytoxan showed that, with appropriate use of 
the drugs, treatment with the combination was 
superior to treatment with either drug alone. 
Urethane, which was inactive in itself, did not 
potentiate the activity of amethopterin, nitrogen 
mustard, or Cytoxan. 6-Azauracil, also inactive 
when employed alone, did not potentiate the ac- 
tivity of 6-mercaptopurine. 

Increasing emphasis has been placed on studies 
of the effectiveness of chemical compounds on 
drug resistance. To date, these have included 
ant i purine- as well as antifolic-resistant variants 
of L1210. Drug- resistant tumors are being used 
to uncover active compounds which may have 
clinical usefulness in cases which have become 
refractory to conventional treatment and to ex- 
plore possible mechanisms of drug action. 

The Sarcoma 37 Assat System. A total of 70 
compounds (26 tested in 1960) has been evaluated 
against early sarcoma 37 for ability to increase 
the life span of the mice. This tumor has been 
particularly insensitive to treatment and has 
therefore not been used routinely in the advanced 
form. Of the compounds tested against early 
S-37, alanine nitrogen mustard, Cytoxan, N- 
methylformamide and p-N, N-bis-(2-iodoethyl) 
aminobenzylphosphonic acid ethyl ester have pro- 
vided appreciable increases in survival time. This 
tumor is of particular interest, because of the ap- 
parent lack of correlation between the degree of 
tumor inhibition and the increase in survival time 
attained as a result of therapy. For example, 
6-mercaptopurine, Bayer 3231, and Actinomycin 
D were able to inhibit local tumor growth in mice 



with sarcoma 37 but provided little, if any, in- 
crease in survival time. 

Special studies employing sarcoma 37 have 
been directed mainly to investigations of the in- 
fluence of the treatment schedule on the effective- 
ness of compounds and to the influence of caloric 
restriction on local tumor growth and survival 
time. 

The Adenocarcinoma 755 Assay System. Un- 
like sarcoma 37, Adenocarcinoma 755 (Ca-755) 
has been quite sensitive to certain compounds, 
particularly the purine antagonists. In earlier 
studies in this program, 45 compounds were 
tested against early Ca-755. Extensive increases 
in survival time as well as tumor-free survivors 
were observed as a result of therapy with 6-mer- 
captopurine and some of its derivatives. The 
high degree of sensitivity of this tumor to ther- 
apy indicated that it could be employed more 
advantageously in an advanced form for the 
quantitative evaluation of drugs. Within the 
past year, 40 compounds have been tested for 
their ability to increase the survival time of mice 
with advanced Ca-755. Many purine derivatives 
elicited substantial increases, but none appeared 
to be more effective than 6-mercaptopurine. 
Among nonpurines, Cytoxan was the only com- 
pound which showed activity in the same range 
as 6-mercaptopurine. Moderate activity was ob- 
served for Actinomycin D and 4-aminopyrazolo 
(3,4-d) pyrimidine. 

Like sarcoma 37, Ca-755 displayed no correla- 
tion between local tumor inhibition and survival 
time extension. For example, three pyrazolo 
(3,4-d) pyrimidines tested produced approxi- 
mately 50 percent inhibition of the local tumor 
of early Ca-755 without providing substantial 
increases in survival time. 

Other tumor systems that might be suitable for 
quantitative assay are under investigation. 

Ehrlich Ascites Tumor. Of eight compounds 
tested against the Ehrlich ascites tumor, only 
N-methylformamide provided an appreciable in- 
crease in survival time. 

Hepatoma 129. Cytoxan and 6-mercaptopurine 
were effective in retarding tumor growth and in- 
creasing survival time of mice inoculated subcu- 
taneously with hepatoma 129 (obtained from Dr. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Belkin), even when the treatment was withheld 
until the tumors reached about 1 cm. in size. 

Moloney Vikus Leukemia. The use of the 
Moloney virus for the production of indicator 
tumors in studies of the chemotherapy of leu- 
kemia in mice which is also being explored, has 
two advantages: (1) the induced neoplasm is a 
host tumor, and (2) the induced disease is similar 
to that which occurs spontaneously in nature. 
Drs. Goldin, J. Glynn and M. Chirigos and 
Messrs. J. M. Venditti and S. It. Humphreys, in 
collaboration with Dr. J. B. Moloney, have evalu- 
ated the therapeutic effectiveness of several drugs 
against this virus-host system, both with virus- 
and whole cell-induced leukemia. Drugs shown 
to be significantly effective in extending the latent 
period (time to death with leukemia) following 
inoculation of neoplastic cells are: Cytoxan, tri- 
ethylenemelamine, and phenylalanine mustard 
(Sarcolysin) ; drugs of intermediate or no effec- 
tiveness are : Amethopterin, dichloroamethopterin, 
6-mercaptopurine, meticortilone and 5-fluoroura- 
cil. With virus-induced indicator tumors, the 
following drugs were moderately effective: Cy- 
toxan, methotrexate, and methylglyoxal-bis- 
guanlyhydrazone. The last-named compound 
produced marked reduction in spleen and thymus 
gland weights. 

Spontaneous Tumors. Because of the poor cor- 
relation between results in animal transplant- 
able tumor screens and in man and because of 
the growing body of information that indicates 
transplantable tumors are usually very different 
from spontaneous ones, screens involving spon- 
taneous tumors need to be established in spite of 
the increase in variability among the experimen- 
tal animals and the longer holding time and 
greater numbers of animals required. Drs. Goldin 
and Chirigos and Messrs. Venditti, Humphreys 
and N. Mantel have initiated studies testing the 
effectiveness of 3',5'-dichloroamethopterin, 6-mer- 
captopurine and Cytoxan on a spontaneous mam- 
mary tumor in strain C3H mice. As might be 
expected, the untreated control animals showed 
considerable variability from day of entry into 
the experiment to the day of death (median, 40 
days; range, 5 to 117 days). Survival time was 



not increased although some retardation of tumor 
growth was observed. The median survival times 
observed suggested that drug toxicity was limit- 
ing with Cytoxan and 6-mercaptopurine in the 
doses employed. 

In spite of logistical difficulties, some data on 
therapeutic effectiveness of several agents against 
spontaneous tumors in noninbred experimental 
animals perhaps should be obtained as another 
comparative screen of importance to clinical 
cancer. 

Biochemical Studies 

3'-Fluoroamethopterin and 3',5'-difluoroameth- 
opterin were tested against leukemia L1210 by 
Drs. A. Schrecker, J. Mead and Goldin and Mr. 
Venditti. The former is comparable in effect to 
methotrexate and the latter to the dichloroanalog. 
These findings suggest that the greater effective- 
ness of 3',5'-dichloroamethopterin over metho- 
trexate in L1210 is not due to steric hindrance 
of the halogen atoms, but to the electronegative 
nature of the halogens. 3',5'-dimethylamethop- 
terin will be synthesized to test further the con- 
cept. 

A simple fluorometric method has been devel- 
oped by Drs. Chirigos and Goldin for the 
determination of small amounts of phenylalanine 
mustard (Sarcolysin), and time studies on the 
distribution of the drug following its administra- 
tion to tumor-bearing animals have been done. 
Binding of the drug by cellular fractions of the 
blood was indicated. 

In collaboration with Dr. N. O. Kaplan (Bran- 
deis University) , Dr. Goldin and Mr. Humphreys 
have studied several precursors, analogs and 
antagonists of diphosphopyridine nucleotide 
(DPN) and related compounds for their influence 
on DPN metabolism, their ability to reverse anti- 
leukemic activity of certain drugs and their anti- 
tumor effects. Dose response curves have been 
obtained on some 50 compounds, with structural 
differences producing a wide range of toxicity. 
Only 6-aminonicotinamide, &ete-acetylpyridine 
and the thiadiazoles have evidenced any antileu- 
kemic effects. Nicotinamide has been shown to 
reverse the antileukemic action of 2-amino-l,3,4- 
thiadiazole and 2-ethylamino-l,3,4-thiadiazole. As 
a follow-up on the earlier finding that nicotin- 
amide administration in the mouse led to more 
than a ten-fold increase in liver DPN, other 



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63 



compounds have been shown also to result in 
rapid formation of liver DPN : 3-acetyl pyridine, 
pyridyl-3-methyl carbinol, fteta-picoline, mono- 
methyl and monoethyl nicotinamide. 

As an example of work representing a continu- 
ation of studies reported last year on the mech- 
anism of action of antifolic drugs, investigations 
are summarized on biochemical studies of resist- 
ance to antifolics in two resistant cell lines (C82 
and M66) derived from leukemia L1210. Drs. 
Schrecker, Mead and Goldin and Mr. Humphreys, 
in collaboration with Dr. M. Friedkin (Tufts 
University) , have found that both the local tumor 
and the spleen of the M66 variant had the same 
amount of dihydrofolic reductase activity as the 
sensitive L1210 leukemia, while in the C82 subline 
the level of enzyme activity was increased thirty- 
fold. Formate-C 14 incorporation into the acid- 
soluble adenine of spleen and local tumor was 
inhibited by antifolics to a much lesser extent 
in the C82 variant than in the sensitive L1210 
line. With single treatment with methotrexate 
or 3',5'-dichloroamethopterin, some inhibition 
was still obtained in the C82 line. With daily 
treatment, however, no inhibition at all was ob- 
served after several days of treatment. This 
would suggest that there are still some sensitive 
cells present in the C82 antifolic-resistant line 
and that they are eliminated early in treatment. 
It tends to support the hypothesis that, in the 
C82 subline, there is a mixed population of cells 
with varying amounts of dihydrofolic reductase, 
the larger proportion of cells having higher 
amounts of the enzyme. The cells that do not 
possess high activity may be the ones eliminated 
during the first few days of treatment. Whether 
in the original L1210 line some cells with high 
dihydrofolic reductase activity are already pres- 
ent has not yet been determined. In the M66 
line, inhibition of formate incorporation by 
methotrexate or 3',5'-dichloroamethopterin (sin- 
gle treatment) is of the same order as in the 
sensitive line. At present the effect of multiple 
treatment in the M66 line is under study. Pre- 
liminary results indicate that, here again, treat- 
ment induces drug tolerance. It would appear, 
so far, that the mechanism of resistance may be 
different in the M66 variant from that in the 
C82 variant. In the latter a high enzyme activity 
produces a high tolerance for the inhibitor. 



Drug Resistance Studies 

Dr. Law continues his studies on the mech- 
anisms involved in inhibition of leukemic cell 
growth by drugs and in development of drug 
resistance. A subline of the lymphocytic neo- 
plasm L1210 has been developed which is resist- 
ant to the effects of 6-mercaptopurine, 5-fluoro- 
uracil and amethopterin. The growth rate and 
strain specificity have become altered. Compara- 
tive studies with the parental cell line on the 
effects of several new agents are in progress. 

With Dr. M. Lane (now at Baylor University) , 
Dr. Law has found that the development of re- 
sistance to 5-fluoropyrimidines apparently has 
changed the sensitivity to cyclophosphoramide 
(Cytoxan) ; "resistant" lines (to three other com- 
pounds) were found to be more sensitive to cyclo- 
phosphoramide than the "sensitive" parental line. 
At optimal dosage levels, complete regressions of 
established tumors in both P-815 and L1210 lines 
have been observed. In collaboration with Dr. 
E. P. Anderson and Dr. W. Brockman (Southern 
Research Institute), strains of both these cell 
lines that are resistant to 5-fIuorouracil were 
found to have decreased capacity (or decreased 
rate) of nucleotide formation. In contrast to 
findings reported by Eeichart, phosphorylase and 
kinase activities, which probably constitute the 
major pathway for the biosynthesis of uridylic 
acid in mammalian tissues, are found in these 
resistant lines. Preliminary results indicate both 
enzyme activities are weaker in the resistant lines 
compared with the sensitive lines. 

Previous studies with intact cells and cell-free 
extracts by Dr. Anderson on the biochemical 
effects of azaserine in azaserine-sensitive and 
azaserine-resistant cell lines of the plasma cell 
neoplasm 70429 indicated an "intact cell factor" 
was operative in the resistance mechanism, but to 
learn if this "factor" was related to greater im- 
permeability of the resistant cells to the drug, it 
was necessary to determine specifically the entry 
of the compound into the cells. In collaboration 
with Dr. J. Jacquez (Sloan-Kettering Institute), 
Dr. Anderson has studied the capacities of the 
two cell lines for concentrative cell transport and 
intracellular breakdown of azaserine. No major 
differences were found in the capacity to concen- 
trate azaserine within the cell. Quantitative dif- 
ferences, however, were found; the sensitive line 
showed a slightly higher initial velocity of up- 



64 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



take, whereas, over a longer period, the resistant 
cells surpassed the sensitive ones in the final 
intracellular concentration which they could 
maintain. No appreciable differences were found 
in the extent of intracellular breakdown of the 
azaserine. Additional studies have been started 
on reversal of azaserine transport by normal 
amino acids in the two lines, since pool size of 
normal metabolites and their capacity to reverse 
transport of the antimetabolites are also impor- 
tant considerations in understanding the mech- 
anism of development of resistance. Some dif- 
ference in pool size of nucleotides has also been 
indicated by data already collected. 

Glycolysis Studies 

In previous Annual Reports, Drs. M. Woods 
and D. Burk reported that a high degree of 
malignancy is associated with greatly increased 
glycolytic capacity and lowered sensitivity to 
glycolytic inhibition of the anti-insulin type. In 
general, those experimental neoplasms in which 
the hexokinase reaction is under strong insulin- 
anti-insulin control respond to one or more 
chemotherapeutic agents better than do those 
cancers in which glycolysis is less readily inhib- 
ited by compounds of the anti-insulin type. 

Human normal and leukemic leucocytes have 
been studied with special reference to their gly- 
colytic responses to anti-insulin steroids, insulin 
and endotoxic polysaccharides by Drs. Woods 
and Burk and Mr. J. Hunter. Results obtained 
to date, though not conclusive, indicate that gly- 
colysis in lymphocytic cells (normal and leu- 
kemic) is much more sensitive to inhibition by 
steroids than is the case with cells of the myelo- 
cytic series. The data indicate that insulin: anti- 
insulin regulation of glucose metabolism plays an 
important role in the metabolism of leukocytes 
(lymphocytic and myelocytic) , and that endotoxic 
polysaccharides counteract such glycolytic inhi- 
bition in a manner resembling, although not iden- 
tical with, insulin. 

During the past year, studies on the action of 
fluorinated pyrimidines and related compounds 
on tumor glycolytic metabolism were extended. 
In collaboration with Dr. J. Seitz (Visiting Sci- 
entist from the Institute for Hematology and 
Blood Transfusion, Leningrad), the effects of 
fluorinated pyrimidines on "high energy phos- 
phate" metabolism of tumor cells were studied. 



At low levels of inorganic phosphate, where in 
vitro glycolytic inhibition may reach 80 percent, 
no reduction in net acid-insoluble high energy 
phosphate was observed. All of this phosphate 
fraction, however, may not have consisted of sim- 
ply ADP and ATP, but might have involved 
possible fluorinated derivatives. 

While inhibitory action by a series of 5-fluori- 
nated pyrimidines seems to depend on the pres- 
ence of an active glucose metabolism during ex- 
posure to the drug, in a few instances, following 
in vivo exposure to 5-fluorouracil, the transplant- 
ability of ascites tumor cells was markedly inhib- 
ited without concomitant glycolytic inhibition. 
However, in all instances, there was a strong 
positive correlation between the in vitro glyco- 
lytic inhibitory activities of a series of substituted 
pyrimidines and their in- vivo anti-tumor ac- 
tivities. 

Evidence was obtained that the anti-glycolytic 
pyrimidines inhibit aerobic glycolysis in ascites 
tumor cells by competitively inhibiting the utili- 
zation of inorganic phosphate during initial 
stages of exposure to the drug. A secondary 
phase of inhibition is not reversed by increasing 
the concentration of inorganic phosphate in the 
medium. A recent report by Lemon is of inter- 
est in this connection: simultaneous intravenous 
administration of glucose with 5-fluorouracil in 
man is accompanied by reduction in host toxicity, 
with retention of anticancer effects. 

Cytoxan, a cyclic phosphoramide derivative of 
nitrogen mustard, was found to inhibit tumor 
cell glycolysis. Comparison of the L1210 ascites 
strain resistant to Cytoxan, developed by Dr. Lane 
with the L1210 ascites susceptible strain, showed 
a difference in the glycolytic and respiratory 
properties of the two strains with respect to 
Cytoxan action. Cytoxan inhibited the glucolysis 
and respiration of the susceptible strain consider- 
ably more than in the resistant strain, the gly- 
colysis and respiration of the tumor being deter- 
mined after action of Cytoxan in vivo. Nicotin- 
amide and diphosphopyridine nucleotide reversed 
Cytoxan action in the resistant more than in the 
susceptible strain. Further progress on the acti- 
vation of Cytoxan in vitro was made. In the 
presence of triphosphopyridine nucleotide (TPN) 
and magnesium, as well as adenosine triphos- 
phate (ATP) and diphosphopyridine nucleotide 
(DPN), Cytoxan inhibition of glycolysis of liver 



NATIONAL CANCER INSTITUTE 



65 



and mammary tumor homogenates and derived 
subcellular elements was obtained. Thus a con- 
nection of Cytoxan action with magnesium and 
TPN was found. An inhibition of ATP-ase by 
5-fluorouracil was demonstrated for the L1210 
ascites tumor cells. Further study of the respira- 
tion and glycolysis of tumors and normal tissues 
was undertaken. Glucose usually produced a 
pronounced Crabtree effect (inhibition of respi- 
ration by glucose) in the case of ascites tumors, 
but this was not always true. TPN, with in- 
creased magnesium and nicotinamide, increased 
the glycolytic production of carbon dioxide three 
to four times above the amount produced in the 
presence of ATP and DPN only, in mouse mam- 
mary tumor homogenates, but not in liver homo- 
genates. An increase in the amount of lactic acid 
produced was also found. This finding has been 
extended to whole cells for the thymoma ascites. 
The increased glycolysis in the presence of TPN 
is not so great with cells, but clearly demonstra- 
ble. There is no evidence to support the hypothe- 
sis that DPN is formed from TPN. Other nu- 
cleosides such as inosine, uridine, cytosine and 
guanosine triphosphates could be substituted for 
adenosine triphosphate partly or wholly in tumor 
glycolysis, in studies with thymoma ascites and 
mammary tumor homogenate. 

Chemotherapy of Experimental Central Nervous 
System Leukemia 

The clinical studies of Drs. E. Freireich and 
L. Thomas in the General Medicine Branch have 
revealed the importance of central nervous system 
involvement as a serious complicating factor in 
chemotherapy of clinical leukemia. Similar 
findings have been seen in mice treated suffi- 
ciently to give extensive increases in survival 
time. An experimental animal model for the 
study of central nervous system leukemia has now 
been developed by Drs. Goldin, Chirigos and 
Thomas and Mr. Humphreys. Treatment with 
amethopterin or 3',5'-dichloroamethopterin in- 
creased the survival times of the mice inoculated 
intracerebrally with L1210 even when the therapy 
was initiated as late as a few days prior to the 
death of the control animals. The effectiveness 
suggests that these drugs may be capable of cross- 
ing the blood-brain barrier in sufficient quantity 



to exert direct antileukemic action in the brain. 
Cytoxan was found to have limited effectiveness 
against intracranial leukemia. 



Service Functions 

The many research contributions made by the 
investigative staff would not be possible without 
the very considerable assistance given by many 
others on the staff of the Institute. Messrs. W. 
Magruder and J. Murphy continue to provide 
invaluable assistance in meeting those many diffi- 
cult day-to-day problems which must be handled 
expeditiously if research progress is to continue 
at a good pace. The skills shown by them are 
not only of tremendous importance to the re- 
search programs, but in a very real sense, often 
require a creativity just as unique as that needed 
for research. 

The administrative clerical and secretarial as- 
sistance provided throughout the Institute, like- 
wise, is in a very real sense a contribution to the 
programs of the Institute without which much 
of the work could not come to fruition. 

The Pathologic Anatomy Branch continues to 
perform very superior work in pathologic anat- 
omy, not only for studies in the NCI but for 
investigations throughout the NHL Pathologic 
anatomy work-up of surgical specimens (2,521 
specimens in 1960) and of autopsy material (276 
autopsies during the year), including the exten- 
sive discussions held by the pathologists with 
other members of the staff, provide basic pathol- 
ogy information of the highest quality for the 
clinical studies under way at NIH. Nearly 50 
scientists in the various laboratories at NIH have 
requested particular tissues from surgical and 
post-mortem services during the past year and, on 
many occasions, it was possible to furnish these 
investigators with fresh human material. 

Six full-time residents in the Department of 
Pathologic Anatomy are receiving excellent ad- 
vanced training in pathology. 

The Cytodiagnostic Service, under the super- 
vision of Drs. R. A. Malmgren and E. "VV. Chu, 
provides excellent exfoliative cytology service to 
NIH and constitutes a major element of the diag- 
nostic facilities available to the clinical staff 
(10,161 slides, representing 3,055 accessions, were 
examined during 1960). The Service has par- 



66 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ticipated in quantitative studies on surgical 
wound washings, on fluids draining from such 
wounds, on body fluids such as spinal fluid, and 
on peripheral blood, utilizing both the Papani- 
colaou staining procedure and special filtration 
techniques for the collection of cells. 

Mr. J. Albrecht and his staff of histopathology 
technicians continued to provide the excellent 
service that the research staff has learned to rely 
on from this superior group. They prepared 
129,068 stained slides for the staff of NIH, of 
which 29,994 required special staining. The fine 



on-the-job training given to technicians, several 
of whom later work closely with members of the 
investigative staff, continues to pay dividends for 
the National Institutes of Health. 

The analytical chemistry group in the Labora- 
tory of Biochemistry under Mr. R. J. Koegel's 
supervision continues to provide high quality 
service. During 1960 the character of the re- 
quests changed significantly, with a reduction in 
requests for microchemical empirical elemental 
analyses, but with an equal workload of some 
5,500 individual analyses. 



NATIONAL HEART INSTITUTE 



INTRODUCTION 

This introduction to a review of the intramural 
research of the National Heart Institute will be 
brief. The material contained in the following 
pages, largely as reported by the leaders of its 
major research groups, speaks for the Heart In- 
stitute far more effectively than can any words 
of introduction. We believe the reports that 
follow combined with the published bibliography 
of the Heart Institute constitute a record of con- 
siderable achievement. That much of it is di- 
rectly pertinent to the categorical responsibilities 
of this Institute will be apparent; we take con- 
siderable pride in the fact that this is accom- 
plished without restriction of the freedom of the 
individual scientist to pursue, within the resources 
available, those problems which excite his intel- 
lectual curiosity. We believe the high level of 
scientific achievement is not separable from this 
freedom. 

Several organizational changes have been ef- 
fected in the Heart Institute. These are not 
reflected in the report which follows since the 
changes were accomplished toward the end of 
the year. The General Medicine and Experimen- 
tal Therapeutics Branch has been for some two 
years an organizational designation without real 
operational meaning. Its three sections, Experi- 
mental Therapeutics, Clinical Endocrinology, and 
Cardiodynamics, have each operated quite au- 
tonomously. This independence has now been 
recognized and the Experimental Therapeutics 
and Clinical Endocrinology groups have been 
given branch status under the leadership of Drs. 
Albert Sjoerdsma and Frederic Bartter, respec- 
tively. The Cardiodynamics Section has been 
combined with the Cardiology Section from the 
Surgery Branch to establish the Cardiology 
Branch under Dr. Eugene Braunwald. That por- 
tion of the Cardiodynamics Section which has 
been the responsibility of Dr. Donald Fry has 
been established as the Section on Clinical Bio- 
physics within the Cardiology Branch. 



LABORATORY OF CELLULAR PHYSIOLOGY 
AND METABOLISM 

Section on Cellular Physiology 

The program of the Laboratory of Cellular 
Physiology and Metabolism, Section on Cellular 
Physiology, is aimed at the understanding of 
what might be called the "design" of protein 
molecules and with the factors concerned in their 
structure, biosynthesis and mechanism of action. 
This broad problem is being attacked from a 
number of directions involving biochemical, ge- 
netic and physical investigations. The major 
premise of the research is that the biological and 
physical properties of proteins are predictable 
from a knowledge of covalent structure alone and 
that much of the mysticism attached to proteins 
has grown out of lack of understanding of pro- 
teins as organic molecules. Thus, it is felt that 
such phenomena as irreversible denaturation and 
the loss of function resulting from treatment by 
such agents as heat, urea, etc., do not necessarily 
reflect irreversible changes in structure, but rather 
ineptitude and lack of information on the part 
of investigators. The following reports of spe- 
cific projects summarize the experimental details 
and future plans of individual segments of the 
program. 

(1) In previously reported studies it has been 
demonstrated that the four disulfide bridges in 
ribonuclease can be cleaved by reduction with 
mercaptoethanol and that the resulting inactive 
product can be converted to the original native 
molecule by simple exposure to atmospheric oxy- 
gen. This work has been extended in the direc- 
tion of the elucidation of the factor concerned 
with the proper pairing of half-cystine residues. 
It is now clear from an examination of the effects 
of a variety of chemical and physical agents that 
the matching of proper half-cystine residues in 
disulfide bond formation is under the control of 
a large number of side chain interactions involv- 
ing hydrogen bonding, electrostatic attractions 



67 



68 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



and repulsions, and Van der Waal's forces. For 
example, when fully reduced ribonuclease, con- 
taining eight sulfhydryl (SH) groups per mole, 
is allowed to reoxidize in the presence of 8 M 
urea, all of these SH groups disappear and a 
tightly coiled spherical molecule is produced 
which is completely inactive in spite of the fact 
that it has physical properties much like those 
of native ribonuclease. Upon re-reduction of this 
substance followed by reoxidation under optimal 
conditions of pll and protein concentration, a 
material which is indistinguishable from the na- 
tive enzyme is obtained in yields up to 95 percent. 
Such an experiment demonstrates the importance 
of hydrogen bonds in the process since urea is 
known to be a strong hydrogen bond rupturing 
agent. The importance of the other interactions 
mentioned above may be demonstrated by the use 
of other substances and conditions. 

Studies of this sort suggest strongly that all 
of the information required for the formation of 
a completely cross-linked and internally coiled 
globular protein is coded into the amino acid se- 
quence of the molecule and that no further infor- 
mation is necessary to direct the formation of 
the complicated three-dimensional structure that 
characterizes this particular enzyme. 

The studies outlined above have involved the 
development of new methods for handling re- 
duced proteins which add considerably to the 
simplicity and reproducibility of the work. It is 
planned during the coming year to apply these 
methods to a variety of other protein molecules 
in an effort to test the generality of the hypothe- 
ses mentioned above. Preliminary findings show 
that another enzyme, lysozyme, behaves in the 
same manner as ribonuclease in relation to the 
reversibility of its reduction and reoxidation. 

(2) Continuing efforts are being made to work 
out the structures, within biologically active pro- 
teins, that are essential for such functional proc- 
esses as catalysis and hormonal stimulation. These 
studies involve specific degradation of ribonu- 
clease and other proteins in an attempt to arrive 
at minimum structures that are still functionally 
competent. It has been possible to mask almost 
all of the positively charged side chain groups 
of ribonuclease by the chemical addition of 
strands of polyalanine to the amino groups of 
the lysine residues without loss in enzyme activ- 
ity or of the ability to regenerate active material 



after preliminary reduction of disulfide bridges. 
It has also been possible to remove several amino 
acids from the carboxyl -terminal end of the en- 
zyme without inactivation although more exten- 
sive degradation has led to complete loss of 
activity. The degradative and reductive experi- 
ments now make possible the preparation of 
fragments of the reduced form of ribonuclease 
which will serve as raw materials for preliminary 
studies on the organic synthesis of portions of 
the protein that might show enzymatic activity. 
Such work is of importance since it is necessary 
to show that those parts of the total structure 
responsible for activity, as indicated by degrada- 
tive studies, are the same as those that will be 
required in the eventual synthesis of an active 
substance. 

(3) Since only certain parts of enzymes ap- 
pear to be essential for function, it seems likely 
that such parts of the structures would be less 
likely to undergo change during speciation and 
evolution in general. If this hypothesis is cor- 
rect, it seems likely that a comparison of the 
same enzyme from different species and from 
mutants of the same species should indicate com- 
mon denominators of structure associated with 
function. Along these lines, a number of differ- 
ent lysozymes are being investigated, both struc- 
turally and with relation to the genetic "infor- 
mation" of the organism in question. The 
structure of lysozyme from chicken egg white 
and from bacteriophage T± are now under active 
investigation and structures of lysozymes from 
a variety of other species of birds are being 
screened in a qualitative way. During the past 
year a chromatographic technique has been per- 
fected in this laboratory which permits the sepa- 
ration of the component peptides of egg white 
lysozyme following reduction and alkylation of 
sulfhydryl groups and trypsin digestion of the 
protein. This technique consists of a gradient 
elution, from phosphorylated cellulose ion ex- 
change columns, using pll and salt gradients of 
a volatile buffer, ammonium acetate. The amino 
acid analyses of the peptides obtained by this 
method provide the background information and 
material necessary for further sequence work. 
Current attempts at specific blocking of selected 
sites during trypsin digestion are aimed at pro- 
viding information which will make it possible 
to assemble the peptides in their proper order. 



NATIONAL, HEART INSTITUTE 



69 



Among the mutants of bacteriophage T4 sev- 
eral have been isolated that produce lysozymes 
with physical properties differing from those of 
the enzyme in the original standard strain of 
phage. The lysozyme of one such mutant has 
been examined in detail and it has been found 
that its structure differs by a change in amino 
acid sequence from that of the standard enzyme. 
Similar examination of other mutants should 
provide information to test the hypothesis that 
the amino acid sequence of an enzyme is deter- 
mined, in a direct fashion, by the sequence of 
genetic "information" coded into the DNA mole- 
cules that determine heredity. The selection for 
interesting enzyme mutants will be continued and 
the fine structure genetic map extended. Ulti- 
mately, it is by the determination of the nature 
of the changes produced in the enzyme as a result 
of mutation (particularly when induced by chem- 
ical mutagens with known mechanisms of action) 
that we hope to gain an insight into the nature 
of the "genetic code." 

(4) Studies on the biochemistry of muscular 
contraction have continued with special emphasis 
on the chemistry of the myosin molecule. In 
earlier studies it was demonstrated that myosin 
is probably made up of three physically and 
chemically identical polypeptide chains, each 
coiled in the form of a helix, the three helices 
being wound together in the form of a rope. 
Present studies now suggest that these three 
strands may be completely dissocated from one 
another and under suitable conditions be made to 
reassociate to yield the original parent molecule. 
The results suggest that the subunit is so de- 
signed that its proper aggregation is predeter- 
mined by the location of interacting groups along 
the chain much in the way that such groups 
determine the re-formation of native ribonuclease 
from the fully reduced molecule as described 
above. The structure of the individual subunits 
is being studied by the preparation of enzymatic 
digests and the separation of the resulting pep- 
tide fragments for subsequent determination of 
sequence. The alignment of these fragments into 
the proper order should eventually yield informa- 
tion on the spacing of interacting groups along 
the chain and may make it possible to explain the 
aggregation process on a rational chemical basis. 
In the course of these studies, a careful determi- 
nation of the molecular weight of myosin by 



light-scattering methods has indicated, in accord- 
ance with the chemical studies, that the native 
molecule is made up of three subunits, each with 
a molecular weight of approximately 200,000. 
These studies have elucidated some earlier ex- 
perimental discrepancies which led to the postu- 
lation of a considerably lower molecular weight 
for this protein. 

It was observed some time ago that the ATPase 
activity of myosin, the protein unit of the con- 
tractile mechanism, exhibits a biphasic response 
to titration of the SII groups, with a 3-4 fold 
stimulation of ATPase activity when approxi- 
mately one-half of the SII groups are titrated, 
and complete inhibition on further titration. The 
initial phase of this process is also characterized 
by a marked alteration in the substrate and acti- 
vation specificities of the protein. During the 
course of the titration, the ability of the molecule 
to react with another muscle protein, actin, is 
lost. This reaction is widely regarded as impor- 
tant in the mechanism of contraction. Identifi- 
cation of the amino acid composition of those 
portions of the molecule involved in these proc- 
esses and their localization in the molecule should 
provide considerable insight into the biochemical 
process of muscular contraction. 

Treatment of myosin with sufficient radioactive 
N-ethylmaleimide to react with one of its sixteen 
sulfhydryl groups leads to a marked acceleration 
of its calcium activated hydrolysis of adenosine 
triphosphate and a complete loss of its ability to 
hydrolyze inosine or guanosine triphosphate. 
Furthermore, the hydrolysis of adenosinetriphos- 
phate is no longer accelerated by ethylenediamine 
tetra-acetate. This process is accompanied by 
the labeling of one of the cysteine containing 
peptides identified by the "fingerprinting" proce- 
dure. In addition, it appears that this peptide 
contains not one but two cysteine residues and 
the titration of this second group occurs during 
the inactivation phase of the biphasic response to 
SH titration. 

(5) Previous studies have indicated that the 
processes of protein biosynthesis in hen's oviduct 
appear to involve the mediation of certain lipid- 
amino acid complexes which are in a very rapid 
state of metabolic flux. The chemical nature of 
these complexes is being studied following their 
partial separation on chromatographic columns. 
It was recently found that specific sites which 



70 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



can distinguish L-valine from D-valine or L-se- 
rine exist in the lipids. Evidence that points to 
a covalent link between the carboxyl group of 
the amino acid and a lipid component was ob- 
tained. Preliminary findings suggest the existence 
of a lipo-peptide thought to be involved in the 
protein synthetic process of the oviduct. 

The lipid-peptide complexes are also being 
studied in E. coli in collaboration with investi- 
gators at the Carnegie Institution of Washington. 
Such cells also contain these materials and the 
results suggest that they may be associated with 
the ribosomes which are known to be actively 
involved in protein biosynthesis. 

In a collaborative project with investigators at 
the National Cancer Institute, chromatographic 
studies are being performed on the ergastoplasm 
of rat liver cells. The ergastoplasm consists of a 
lipid membrane, studded with ribonucleoprotein 
granules approximately 150 A in diameter. It is 
believed that "information" for making particu- 
lar proteins is contained in the configuration of 
the nucleic acids of these granules. If this hy- 
pothesis is correct, one would expect the granules 
to be heterogeneous. The granules are being frac- 
tionated to determine if different granules lead 
to the synthesis of different cytoplasmic proteins. 

(6) Studies on the structure and metabolism 
of heparin and related mucopolysaccharides, the 
relation of heparin to lipoprotein lipase and the 
role of lipoprotein lipase in fat metabolism are 
being continued. Olive oil, corn oil, cream and 
cocoa butter were fed to rats with cannulated 
thoracic ducts. The chyle was collected and 
chylomicrons prepared from it. These chylomi- 
crons were then incubated with pancreatic lipase 
and lipoprotein lipase. The free fatty acids lib- 
erated by enzymic hydrolysis were isolated and 
the molar percentage composition determined by 
gas-liquid chromatography and compared to that 
of the substrate triglyceride. The relative 
amounts of free fatty acid and glycerol produced 
during hydrolysis were also determined. The re- 
sults show clearly that, whereas pancreatic lipase 
hydrolyzes preferentially fatty acids esterified at 
the a-position of a triglyceride, lipoprotein lipase 
hydrolyzes a- and ^-esters equally well. Lipopro- 
tein lipase does not appear to have a specificity 
among the long chain fatty acids, saturated or 
unsaturated. 



Section on Metabolism 

The Metabolism Section of the National Heart 
Institute has a wide range of current interests 
which center primarily on the chemistry and 
metabolism of two classes of compounds, lipids 
and proteins. The two are in many respects in- 
terrelated but the study of lipids is perhaps para- 
mount and can be considered as part of an overall 
program aimed at elucidating the causes of 
atherosclerotic heart disease. 

Metabolic Activity of Adipose Tissue 

One of the major projects in the laboratory in- 
volves the study of the metabolic functions of 
adipose tissue and their control. This has been 
approached experimentally in several ways. The 
mechanisms through which the rate of fatty acid 
release is regulated and altered by hormones is 
of primary interest and several kinds of experi- 
ments have been carried out to explore the hy- 
pothesis that alterations in the rate of fatty acid 
release may result from changes in the rate of 
fatty acid esterification. 

Triglycerine Synthesis. Glycerides are syn- 
thesized from fatty acyl Coenzyme A and ^glyc- 
erophosphate by a mechanism apparently like that 
previously described in liver. The adipose tissue, 
however, cannot convert glycerol to ^glycero- 
phosphate as can some other tissues. The syn- 
thesis of a-glycerophosphate from other metabolic 
interemediates was studied in the homogenate 
system. Synthesis from phosphorylated interme- 
diates of the glycolytic cycle was obtained when 
DPNH was added to the system. Neither glucose 
nor glycogen was an effective precursor in this 
system. Synthesis of triglyceride from diglycer- 
ide and acyl Co-A precursors has been observed 
in chicken adipose tissue, but the excessive thio- 
lase activity of rat adipose tissue has prevented 
successful studies in that species. One interesting 
aspect of the studies of incorporation of fatty 
acids into glycerides is that lipolysis continues, 
even though net esterification of fatty acids is 
occurring. Characteristics of the lipase active 
in this in vitro system suggest that it is different 
from lipoprotein lipase. 

Influence of Hormones. It has been shown 
that epinephrine, ACTH, and glucagon (which 
increase fatty acid release) all decrease the in- 



NATIONAL HEART INSTITUTE 



71 



corporation of C 14 -palmitate into glycerides by 
adipose tissue incubated in vitro. Serotonin, 
which does not increase fatty acid release, does 
not alter C 14 -palmitate incorporation; while glu- 
cose, which stimulates palmitate esterification, 
decreases fatty acid release. The effects of these 
hormones and of a series of compounds related 
to epinephrine on the conversion of C 14 -glucose 
to triglyceride have also been studied in an at- 
tempt to elucidate the interrelationships between 
carbohydrate and lipid metabolism in adipose 
tissue. Some similar studies have been carried 
out in tissues obtained from rats with altered 
thyroid function, comparing effects in these tis- 
sues to those obtained in tissues from normal 
animals in order to determine the role of thyroid 
state in the responses of adipose tissue. 

Physiology of Adipose Tissue and the Mechanism 
of Free Fatty Acid Release 

Fatty acids released by adipose tissue are car- 
ried through the plasma in association with 
serum albumin. The equilibrium relationships 
between fatty acids and albumin were studied in 
1957 and 1958 by Dr. DeWitt Goodman. During 
1960 an attempt was made to measure with a 
stop-flow apparatus the kinetics of the formation 
of the fatty acid albumin complex. The only 
conclusion possible was that the reaction is com- 
plete in so short a time that this apparatus is 
not satisfactory for a quantitative measurement. 
It may be concluded that the formation and dis- 
sociation of fatty acid albumin complex requires 
a time that is short in relation to the time re- 
quired for blood plasma to pass through a capil- 
lary in vivo. Further preliminary experiments 
aimed at measurements of the diffusion of fatty 
acids through albumin containing media were 
undertaken. These indicated that fatty acids 
ions cannot diffuse appreciably faster than can 
the albumin to which they are attached. 

Biosynthesis of Cholesterol 

A major interest of one group in the laboratory 
has been the mechanism of action of MER-29. 
This compound has been found to affect experi- 
mental animals and man in the same way — 
namely, by inhibiting the reduction of the side 
chain of 24-dehydrocholesterol to yield choles- 
terol. As a result, the serum of patients treated 
with MER-29 contains a considerable quantity 



of desmosterol (24-dehydrocholesterol), which 
may be as much as y± of the total sterol present. 
It also accumulates in rat tissues and presumably 
in man as well. Desmosterol appears to partici- 
pate in many of the metabolic reactions of choles- 
terol and is found to be esterified at much the 
same rate. Whether it may give rise to bile acids 
and steroid hormones in which the side chain is 
lost is the subject of a current investigation. 
Desmosterol yields less color in the standard 
Liebermann-Burchard color reaction than does 
cholesterol, with the result that the application 
of standard cholesterol methods to MER-29 
treated patients results in a falsely low estimate 
of serum sterol levels. Colorimetric and chrom- 
atographic methods for the estimation of true 
cholesterol and desmosterol levels have been de- 
veloped and publications currently in preparation 
or in press emphasize these data. 

One of our investigators has had the privilege 
of working with Dr. George Popjak, both in 
London and during Dr. Popjak's tenure as a 
Visiting Scientist at NHI. Their collaborative 
work dealt with studies of the mechanisms of 
biosynthesis of some of the important intermedi- 
ates in the pathway to cholesterol. It was found 
that both liver microsomes and a soluble enzyme 
were required for the cyclization of squalene. 
2 and TPNH were also essential. Studies with 
inhibitors indicated the essential participation of 
enzyme sulfhydryl groups in the reaction. The 
mechanism for the conversion of f arnesyl pyro- 
phosphate to squalene was also studied. The 
available evidence indicates that the mechanism 
of this condensation is asymmetric, one farnesyl 
pyrophosphate being converted to an intermediate 
which differs from that to which the other far- 
nesyl pyrophosphate is changed. There have been 
important technical difficulties with this study 
which have made it impossible to define the de- 
tails so far but the study is being actively pursued 
at this time. 

Hypoproteinemia and Hyperlipemia of Nephrosis 
Clinical studies of nephrosis which were pur- 
sued in previous years have been curtailed since 
the departure of Dr. Howard Goodman from this 
Laboratory but experimental animal work has 
continued. Plasmapheresis has been utilized to 
simulate the renal loss of proteins. This proce- 
dure results in consistent and significant increases 



72 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in serum lipids. The mechanism of this effect is 
under current investigation. The relation of car- 
bohydrate feeding and infusion to serum triglyc- 
eride levels is also being scrutinized. Earlier 
experiments both in animals and in patients led 
to the finding that a high intake of carbohydrate, 
while decreasing the serum FFA level often led 
to an increase in serum triglycerides. 

Relation of Serum FFA Levels to Lipid Deposi- 
tion in Tissue 

In a study that was completed during 1960 and 
has been prepared for publication, investigators 
of the Metabolism Section demonstrated that the 
sustained elevation of FFA levels following 
norepinephrine infusion was correlated with an 
increased deposition of triglyceride in the livers 
of dogs. Research in other institutions has indi- 
cated that the uptake of FFA by tissues (heart 
and liver) is related to arterial FFA levels. 
These various findings add further conviction to 
the conclusion that FFA transport is regulated 
largely by the adipose tissue that controls its 
release into the blood rather than by tissues that 
utilize it. 

Clinical Studies of Hypoproteinemia 

In this study, carried out in collaboration with 
.the Metabolism Section of the National Cancer 
Institute, patients with various types of hypo- 
proteinemia have been admitted and studied. 
Two patients with analbuminemia have been en- 
countered. Each of these was given S 35 -labeled 
amino acids and studied for evidence of incorpo- 
ration into serum proteins. Surprisingly, the 
serum albumin fraction, however carefully puri- 
fied, contained some radioactivity. This result 
suggests that each subject makes at least a small 
amount of endogenous serum albumin and agrees 
well with the report from Bennhold's clinic that 
analbuminemics, who have never been given albu- 
min, have traces of this protein in their plasma. 

A much larger group of hypoproteinemic pa- 
tients has been shown to be suffering from "pro- 
tein losing enteropathy." This condition is dem- 
onstrated by testing with I 131 PVP and I 131 
albumin. Clinical and biopsy studies of this group 
of patients have led to the definition of a pre- 
viously unrecognized disorder of the intestinal 
lymphatics, for which the term "intestinal lym- 
phangectasia" is being proposed. The results of 



this study were presented at a meeting of the 
American Gastroenterological Association and are 
being readied for publication. I 131 PVP has 
been prepared regularly with the help of the 
Clinical Center Pharmacy Department in such 
quantity that it has been possible to provide it 
to investigators in other parts of the country and 
of the world. This program has resulted in the 
discovery of a large number of similar cases in 
other centers and to the appearance of several 
additional publications, primarily in foreign 
journals. 

Fibrinogen 

An investigation into the basic structure of 
fibrinogen has been continued during 1960. The 
amino acid composition of this protein has been 
determined with exactness and the products of 
chemical and enzymatic hydrolysis are under 
study. The complexity of fibrinogen makes it 
unlikely that the complete amino acid sequence 
will be known soon but some important facts 
have emerged. Trypsin attacks fibrinogen and 
quickly degrades it into a few large fragments. 
The most important fragment has been isolated 
and appears to have a molecular weight of ap- 
proximately 100,000. This may be equivalent to 
the spherical portions of the fibrinogen molecule 
seen in electron micrographs. The relation of 
fibrinogen structure to its role in the clotting 
mechanism is a basic interest in this work. 

Fatty acid transport 

Experiments have been designed this year to 
eliminate certain irrelevant biological variation 
when the metabolism of different fatty acids is 
being compared. This is accomplished by intro- 
ducing simultaneously such acids as C 14 -labeled 
linoleic acid and HMabeled palmitic acid. These 
have been fed or injected as free fatty acids 
(FFA) or chylomicron triglyceride fatty acids 
(TGFA). The results so far have revealed some 
hitherto unappreciated differences in the rates of 
removal from the plasma of these two acids as 
FFA. This is detected by an early drop in the 
ratio of labeled linoleic to palmitic in plasma. 
This, in turn, is explained in part by a very rapid 
preferential accumulation of linoleic acid by the 
lecithin in liver. A greater accumulation of 
palmitic by the liver triglycerides is not sufficient 
to overcome the effect on the apparent plasma 



NATIONAL HEART INSTITUTE 



73 



removal rates. This technique shows promise of 
permitting comparative measurements not other- 
wise available in events occurring as fast as the 
turnover of fatty acids. 

Means of interpreting such experiments using 
tracer fatty acids have been improved by the 
programming of data obtained in animal and 
human experiments, such as the departure of 
radioactivity from the plasma or its appearance 
in the expired air, for the 650 computer. To 
this has recently been added electronic means of 
resolving sums of exponentials into components, 
a system developed in LTD-NHI. Combining 
these operations, it is possible with a few mo- 
ments of calculation to obtain expressions for 
the probability of conversion of a fatty acid to 
carbon dioxide or of its recycling within the 
plasma lipid compartment. Presently under study 
is the question of how useful such operations are 
in interpreting biological data of this rather 
standard type, and whether unique information 
can be so obtained. 

Regulation of Lipoprotein Synthesis 

The regulation of plasma lipid and lipoprotein 
levels has been studied both from the standpoint 
of the lipid and the protein moieties and from 
differing experimental approaches. 

Cholesterol Catabolism. The action of dietary 
fats on cholesterol metabolism was measured by 
using the turnover of bile acids as a sensitive 
indicator of cholesterol degradation. Preliminary 
data suggest that cholic acid turnover may be 
influenced by dietary fat, but final analyses are 
awaited for more certain interpretation. Studies 
of cholesterol degradation using measurement of 
the rate of appearance of end products of labeled 
cholesterol in feces have failed to demonstrate 
a significant correlation with the nature of the 
dietary fat. 

Rate of Lipoprotein Synthesis In Vitro. It 
having been previously demonstrated in the labo- 
ratory that liver slices synthesized the protein 
moiety of serum lipoproteins, and that the rate 
of such synthesis was not influenced by factors 
increasing cholesterol synthesis, elaboration of 
other lipids in this system was studied. It has 
been observed that the presence of high concen- 
trations of free fatty acids in the medium sig- 



nificantly increases the rate of triglyceride syn- 
thesis by the liver. Concomitant effects of glu- 
cose and of epinephrine were consistent with the 
overall hypothesis that increased mobilization of 
free fatty acids from adipose tissue to the liver 
could increase liver triglyceride synthesis and 
ultimately lead to higher levels of triglyceride 
and other lipids, and hence lipoproteins, in 
plasma. 

Epinephrine-indtjced Htperlhtdemia. Epi- 
nephrine or norepinephrine were infused intra- 
venously into anesthetized dogs. Rises in serum 
free fatty acids in all dogs were accompanied by 
large increases in liver triglyceride concentrations. 
Intraportal infusion of norepinephrine, which by- 
passed the effect of adipose tissue, did not change 
liver triglycerides. This, and the fact that the 
total content of lineoleic acid in the liver rose 
(despite the fact that this acid cannot be syn- 
thesized by the dog) indicate that the fatty liver 
must have arisen because more lipid was mobilized 
from the adipose tissue via the FFA pathway 
than could be oxidized. The above effects were 
associated with some rise in serum cholesterol, but 
not triglycerides. It is possible, thus, to relate 
influences, such as stress, to rises in serum lipo- 
proteins, through the mobilization of FFA. This 
mechanism has considerable importance in under- 
standing a number of currently vague influences 
upon plasma lipid levels. 

Defective Lipid Metabolism 

Plasma Post-Heparin Lipolytic AcnvrrY. 
There are presently no biochemical tests for 
segregating metabolic defects which may cause 
"essential hyperlipemia." Despite many studies 
of lipoprotein lipase activity, no attempt has been 
made to use a quantitative measurement of the 
activity of this enzyme in man after heparin ad- 
ministration as a means of such segregation. Such 
an enzyme assay has been developed, a set of 
optimal conditions determined, and rates of 
lipolysis established in a group of 25 normals, 
and these compared with subjects with hyper- 
lipemia. The latter show great scatter, falling 
into three vaguely defined groups, equal to, lower, 
or higher than normal. More importantly, it was 
discovered for the first time, that the response to 
heparin in man varies with the previous diet. 



74 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Low fat intake for 7-10 days depresses enzyme 
activity to about half of normal. It is also inter- 
esting that normal individuals with such de- 
pressed activity do not have hyperlipemia even 
though their enzyme levels, as measured in vitro, 
are lower than those in some patients with severe 
familial hyperlipemia. 

A New Lipidosis. An apparently new disorder 
of lipid metabolism has been discovered in two 
siblings of a family from the Island of Tangier 
in Chesapeake Bay. It is manifested by tonsillar 
enlargement and discoloration, lymphadenopathy, 
and enlargement of liver and spleen. Such 
changes in the reticuloendothelial tissues are asso- 
ciated with accumulation of foam cells contain- 
ing lipid. Practically all the excess lipid appears 
to be cholesterol esters. The pedigree has been 
established and many relatives examined during 
a trip to Tangier Island. The paternal and ma- 
ternal grandfathers five generations removed in- 
clude brothers and the pedigree is theoretically 
typical for the manifestation of the disease as the 
homozygous phenotype of a rare recessive gene. 
Whether a heritable defect in lipid metabolism is 
responsible remains to be proved. Studies in 
process to elucidate the mechanisms include ton- 
sillar incubations with cholesterol precursors and 
measurement of plasma cholesterol turnover in 
both children. One fortuitous finding which is 
being further explored is the potential value of 
the tonsil for study of lipid synthesis in the RE 
system. It is a relatively available tissue, and 
slices can be easily prepared which readily incor- 
porates precursors into cholesterol. 

Section on Enzymes 

The activities of the Section on Enzymes are 
directed toward the elucidation of the detailed 
mechanisms by which various compounds are 
metabolized in living organisms. Attention is 
directed to the metabolism of those substances 
which is poorly understood and which offers a 
unique opportunity to study biochemical phe- 
nomena of fundamental interest and general sig- 
nificance. Specifically, the current research in- 
cludes studies on various aspects of lipid metabo- 
lism, amino acid metabolism, the metabolism of 
heterocyclic compounds, of onium compounds, 
and of one carbon compounds. It also includes 



studies on complex biochemical phenomena such 
as the regulation of cellular metabolism, enzyme 
induction, and cellular differentiation. 

Lipid Metabolism 

Fatty Aero Synthesis in C. kluyveri. It was 
previously reported that cell-free extracts of C. 
kluyveri catalyze the incorporation of C0 2 into 
the unesterified carboxyl group of malonyl-CoA; 
this exchange reaction requires the presence of 
malonyl CoA and an acyl CoA derivative of a 
saturated fatty acid with 2-16 carbon atoms in 
addition to C0 2 . 

The cell-free extract of C. kluyveri has now 
been separated into two soluble partially purified 
protein fractions both of which are necessary for 
the exchange reaction. Although the mechanism 
has not been fully elucidated, evidence has been 
obtained suggesting that the exchange reaction 
involves the reversible condensation, with con- 
comitant decarboxylation, of malonyl CoA and 
the acyl CoA derivative to form a /?-keto thiol- 
ester derivative which is firmly bound to the 
enzyme : 

COOHCH 2 COSCoA + RCOSCoA + E ?* 
E-RCOCH 2 COSCoA + C0 2 + CoASH 

The likelihood that this reaction represents the 
first step in the biosynthesis of long chain fatty 
acids is supported by the observation that a com- 
pletely soluble enzyme complex precipitating be- 
tween 65-95 percent saturation with ammonium 
sulfate catalyses the synthesis of stearate, C-16, 
C-20 and C-22 fatty acids from the same reac- 
tants, namely, acetyl CoA, malonyl CoA and 
TPNH. 

Fatty Aero Synthesis by Adipose Tissue. Sub- 
microsomal particles derived from epidydimal fat 
pads of the rat have been found to contain an 
enzyme system capable of synthesizing palmitate 
and small amounts of stearate from acetyl CoA, 
malonyl CoA and TPNH. The stoichiometry of 
the synthesis of palmitate can be expressed as 
follows : 

Acetyl-SCoA + 7 malonyl-SCoA + 14TPNH + 

14H+ ->► palmitate + 7C0 2 + 8C0ASH 

+ 14TPN. 

The overall reaction proceeds most rapidly at 
pH 6.8 and is activated by mercaptans. 



NATIONAL HEART INSTITUTE 



75 



As with the bacterial enzyme preparation, the 
rat fat pad enzyme system was found to catalyze 
the interchange of C0 2 and the carboxyl group 
of malonyl CoA. The observation that the ex- 
change reaction and the fatty acid synthesizing 
activity of the fat pad enzyme concentrated in 
parallel throughout a 60-fold purification pro- 
vides additional support for the conclusion that 
the CO2 exchange reaction is an integral part of 
the system required for fatty acid synthesis. A 
study of the specificity of the fatty acid synthe- 
sizing enzyme system for fatty acid acyl CoA 
derivatives indicated that odd numbered and 
branched chain derivatives could substitute for 
acetyl CoA. In such cases, odd numbered, iso, 
and anteiso long chain fatty acids were formed. 

Incidental to this investigation has been the 
development of procedures for the non-enzymatic 
synthesis of /?-keto acyl CoA derivatives and for 
the isolation, separation and quantitation of long 
chain fatty acids as their hydroxamate deriva- 
tives. 

Propionate Metabolism. The roles of biotin and 
vitamin B 12 coenzymes in the metabolism of pro- 
pionate have now been established in collabora- 
tion with P. Overath, H. Eggerer and F. Lynen 
of the Max-Planck Institute, Munich, Germany. 
In studies with enzyme preparations of Propioni- 
bacterium shermanii it was shown that biotin 
which is enzyme bound (E* Biotin) serves 
as the CO2 acceptor in a reaction equilibrating 
propionyl-CoA with methylmalonyl-CoA as fol- 
lows: 

methylmalonyl-CoA + E* Biotin «=s Co 2 'E* 
Biotin + Propionyl-CoA. 

The COvE* Biotin is presumed to be the car- 
boxyl donor for the conversion of pyruvate to 
oxalacetate in this organism (Swick and Wood, 
1959) and may react also with ADP and Pi to 
produce Co 2 , ATP and regenerate E # Biotin 
(Ochoa, et al.) . Vitamin Bi 2 coenzyme was iden- 
tified as a cocatalyst for the reversible isomeriza- 
tion of methylmalonyl-CoA to succinyl-CoA. 

Investigations of the mechanism of enzymatic 
isomerization of methylmalonyl-CoA to succinyl- 
CoA have been made. Studies on this reaction 
have been hampered by lack of any model reac- 
tion in organic chemistry. Recently, however, the 
rearrangement of a-cinenic acid to geronic acid 



has been shown to involve a carboxyl-group trans- 
fer. The further demonstration that the rear- 
rangement was actually a transcarbonylation, 
mediated by elimination of carbon monoxide and 
its recapture by a resonance-stabilized carbonium 
ion, has prompted a reinvestigation of the methyl- 
malonyl-CoA isomerase mechanism, particularly 
since the B^ coenzyme for the enzymatic reaction 
could serve as a carbon monoxide carrier through 
its cobalt moiety. With lamb kidney enzyme 
preparations entirely dependent on the addition 
of B 12 coenzyme a search was made for formation 
of C 14 from succinyl-1, 4— C 14 -CoA, or its incor- 
poration into the latter in the presence of methyl- 
malonyl-CoA. The results were negative. 

In other studies with partially purified isomer- 
ase from P. shermanii it was demonstrated that 
the isomerization of 2-C 14 -labeled methylmalonyl- 
CoA yields 3-C 14 -labeled succinyl-CoA. Thus, it 
is established that the isomerization involves a 
migration of the thiolester carbon atom to the 
^-methyl carbon of methylmalonyl-CoA and elim- 
inates from consideration the idea advanced by 
others that the isomerization involves intermolec- 
ular transcarboxylation reactions. 

Metabolism of Onium Compounds 

Anaerobic Fermentation of Choline. Acetalde- 
hyde has been identified as an intermediate in the 
anaerobic conversion of choline to trimethylamine, 
acetate and ethanol by Vibrio cholinicus. The 
latter two substances are derived from the dis- 
mutation of acetaldehyde which is catalyzed by 
alcohol and acetaldehyde dehydrogenases present 
in the extract. This dismutation is associated 
with the esterification of inorganic phosphate. 
The alcohol dehydrogenase was purified 65-fold 
and was demonstrated to have an absolute re- 
quirement for TPN. The aldehyde dehydrogen- 
ase has been purified about 20-fold; it is TPN 
specific and is maximally activated by addition 
of CoASH. These observations, and the presence 
in cell-free extracts of phosphotransacetylase and 
acetokinase, suggest that phosphate esterification 
associated with acetaldehyde dismutation involves 
the intermediary formation of acetyl-CoA and 
acetyl-P, followed by a transfer of the phosphoryl 
group from the latter compound to ADP with the 
formation of ATP and acetate. This phosphate 
esterification is not inhibited by dinitrophenol ; it 



76 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



therefore probably accounts for the dinitrophenol 
insensitive portion of the total phosphate esteri- 
fied during the fermentation of choline by cell- 
free extracts. 

A new organism belonging to the genus Clos- 
tridium and capable of fermenting choline anaero- 
bically has been isolated. Although considerable 
variation in the composition of the fermentation 
products is observed with different cultures, acet- 
aldehyde, acetate, ethanol, trimethylamine and 
ammonia have been identified as major products. 
The formation of ammonia is particularly signifi- 
cant since it must reflect a considerable metabo- 
lism of the tertiary amino group under anaerobic 
conditions. The fate of the methyl groups is under 
investigation. 

Sulfontum Compounds. In an effort to deter- 
mine if the energy associated with the hydrolysis 
of the sulfonium bond (-21,000 cal.) can be used 
for cellular metabolism, an anaerobic organism 
capable of growing on dimethyl-/?-propiothetin as 
the major source of carbon has been isolated in 
pure culture from soil by the enrichment culture 
technique. The fermentation balance for the 
degradation of propiothetin may be tentatively 
written as follows : 

+ 
3(CH 3 ) 2 -S-CH 2 CH 2 COOH + 2H 2 ^ 
2CH 3 CH 2 COOH + 3(CH 3 ) 2 S + C0 2 + 
3H+ + CH3COOH 

The possibility that this fermentation involves a 
primary cleavage of propiothetin to dimethyl 
sulfide and acrylate which is followed by a sec- 
ondary fermentation of the acrylate to propionate, 
acetate and C0 2 is indicated by the fact that cell 
suspensions are capable of fermenting acrylate to 
the latter products. 

It has been established that cell-free extracts 
are capable of catalyzing the fermentation of 
propiothetin but the products have not been de- 
termined. 

Incidental to this investigation has been the 
development of a new sensitive analytical method 
for the determination of dimethyl-propiothetin. 

Metabolism of Heterocyclic Compounds 

Riboflavin' Degradation. It was previously 
shown that the oxidative dissimilation of ribo- 



flavin by an aerobic organism involves the inter- 
mediary formation of l-ribityl-6,7-dimethyl-l,2, 
3,4-tetrahydro-2,3-diketoquinoxaline. This com- 
pound is subsequently converted to 6,7-dimethyl- 
2,3-quinoxalinediol by an oxygen dependent 
cleavage of the N-ribityl bond. In order to fa- 
cilitate further studies on the nature of the cleav- 
age reaction and to establish the fate of the 
ribityl moiety, l-ribityl-6,7-dimethyl-l,2,3,4-tetra- 
hydro-2,3-diketoquinoxaline-l'-C 14 was prepared 
by a non-enzymatic synthetic procedure. The en- 
zymatic degradation of this labeled intermediate 
is currently under investigation. 

Plausible intermediates in the further enzy- 
matic conversion of 6,7-dimethyl-2,3-quinoxaline- 
diol to 3,4-dimethyl-a-pyrone-6-carboxylic acid 
and oxamide are 6,7-dimethyl-2,3,5-quinoxa1ine- 
triol and 3,4-dimethyl-a-pyrone-6-carboxylic oxa- 
mide. Attempts to synthesize the latter sub- 
stances have been made, but as yet the desired 
compounds have not been obtained. 

In order to simplify further studies on the 
organism responsible for riboflavin degradation, 
the factors present in yeast extract and peptone 
required for its growth have been determined. It 
has been established that methionine, lysine, 
threonine, valine, isoleucine and tyrosine are all 
needed for maximum growth. It was further 
determined that the degradation of riboflavin is 
an inducible metabolism in this organism and 
that ribose but not ribitol will serve as an energy 
source for growth. 

Biosynthesis of Phenazine-1-Carboxtlic Actd. 
The ability of various labeled metabolites to serve 
as a source of carbon for the biosynthesis of 
phenazine-1-carboxylic acid by washed cell-sus- 
pensions of Pseudomonas aureofaciens was inves- 
tigated. The isotopic carbon from formate-C 14 , 
acetate-1-C 14 , lactate-1-C 14 , alanine-2-C 14 , glyc- 
erol-l,3-C 14 , threonine-U-C 14 , glucose-6-C 14 , pyru- 
vate-l-C 14 , serine-3-C- 14 , tyrosine-U-C 14 , and 
tryptophan-3-C 14 is readily incorporated into the 
phenazine pigment. On the other hand isotope 
from glucose-1-C 14 was not incorporated. Efforts 
to demonstrate a common precursor role for some 
of these substances by means of the isotopic com- 
petition technique were unsuccessful. Thus, the 
extent of isotope incorporation with glycerol -1,3- 
C 14 was uninfluenced by pools of unlabeled serine, 



ah 



NATIONAL HEART INSTITUTE 



77 



alanine, or glycine. Efforts to obtain mutant 
strains blocked in their capacity to synthesize the 
phenazine pigment were also unsuccessful. Inhi- 
bition of pigment formation has been observed 
with various aromatic compounds such as o-anisi- 
dine, aniline, o-phenylenediamine, anthranilic 
acid, etc. 

Regulation of Biosynthetic Pathways 
Aspartate metabolism. Eegulation of diverse bio- 
synthetic pathways by changes in concentrations 
of the ultimate products is achieved by two dis- 
tinct mechanisms: (1) a repression of the synthe- 
sis of enzymes involved in the metabolic pathway, 
and (2) specific inhibiation of the activity of the 
first enzymatic step in the metabolic sequence. 
The first mechanism has been referred to as "re- 
pression" and the second is known as "feedback" 
control. 

In collaboration with G. N. Cohen of the Pas- 
teur Institute, Paris, France, it has been shown 
that in E. coli aspartokinase catalyzes the first 
step in a metabolic sequence leading ultimately 
to the formation of the three different amino acids 
threonine, lysine and methionine. Its control by 
either of the above regulatory processes therefore 
presents a unique situation in which excessive 
production of one of these amino acids could lead 
to decreased aspartokinase activity and conse- 
quently to a deficiency in the production of the 
other two amino acids. 

Evidence has been obtained showing that in 
E. coli a partial solution to this dilemma is ob- 
tained through the synthesis of two different 
aspartokinases which are independently controlled 
by the concentrations of L-lysine and of L-threo- 
nine. One enzyme is specifically and noncompeti- 
tively inhibited by L-lysine and its formation is 
completely repressed when the organism is grown 
in the presence of 10~ 2 M L-lysine. The other 
aspartokinase is specifically and competitively in- 
hibited by L-threonine but its concentration is 
not significantly influenced by growth in the pres- 
ence of L-threonine. The biological implications 
of this discovery are supported by the further 
observation that in yeast, where the synthesis of 
lysine does not involve aspartate as a precursor, 
the lysine sensitive aspartokinase is virtually ab- 
sent; here the threonine sensitive enzyme is the 
major if not the only enzyme present. 



Metabolism of Amino Acids 



Conversion of Phosphohomoserine to Threo- 
nine. The mechanism previously proposed for 
this unique transformation in which alcohol isom- 
erization is coupled with phosphate elimination 
has now been confirmed. When this reaction is 
carried out in 100 percent D 2 0, exactly 2 solvent 
hydrogen atoms appear in newly formed threo- 
nine. A complete hydrogen degradation of threo- 
nine has been developed, and has shown that in 
all cases solvent hydrogen is incorporated only 
into the a and /? positions. The small incorpora- 
tion of solvent H 3 previously reported reflects 
addition of protons to double bond 40 times faster 
than tritium ions. The study of the mechanism 
of this unique reaction is still at an early stage. 
While the coenzyme (pyridoxal-P) can be formu- 
lated as potentiating removal of phosphoric acid, 
shifting of double bonds, and oriented addition 
of water, neither it nor phosphate can as yet be 
invoked to account for the different products in 
the reactions: homoserine -> a-ketobutyrate, 
phosphohomoserine ->- threonine. 

Sulfur Transfer Between Homocysteine and 
Cysteine. In an effort to elucidate the detailed 
mechanism whereby the reversible transfer of sul- 
fur from homocysteine to cysteine occurs in intact 
microorganisms, and in order to explain the non- 
reversibility of this process in mammals, the 
metabolism of cystathionine has been investigated 
at the enzyme level. An enzyme catalyzing the 
cleavage of cystathionine to a-ketobutyrate, cys- 
teine and ammonia has been purified 800-fold 
from extracts of a Neurospora mutant believed to 
lack the ability to form cysteine and homocys- 
teine. More slowly this enzyme catalyzes the 
conversion of homocystein to a-ketobutyrate and 
ammonia and of cysteine to pyruvate and am- 
monia. Qualitative differences between it and 
the homologous crystalline enzyme from liver are 
already apparent. 

Y-Aminobutyric Aero Metabolism. It was pre- 
viously established that extracts of Clostridium 
aminobutyricum catalyze the conversion of y-ami- 
nobutyrate to y-hydroxybutyrate and NH 3 . This 
conversion involves the intermediary formation 
of succinic semialdehyde by a mechanism which 
is obligately linked with transamination to a-keto- 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



glutarate (needed in catalytic amounts) followed 
by DPN-dependent oxidative deamination of the 
glutamate formed. This is coupled with the re- 
duction of succinic semialdehyde to y-hydroxy- 
butyrate. The further metabolism of y-hydroxy- 
butyrate to acetate and butyrate by dialyzed 
extracts requires orthophosphate, DPN, TPN and 
acetyl CoA : presumably, the acyl CoA derivatives 
of vinyl-acetate, crotonate, /?-hydroxybutyrate 
and acetoacetate are involved as intermediates. 

Elsden and his associates have shown that 10 jug 
of dry bacteria are produced for each /unole of 
ATP formed during the anaerobic fermentation 
of various substances by organisms of various 
kinds. In the present study it has been found 
that the fermentation of y-hydroxybutyrate and 
of y-aminobutyrate by C. aminobutyricum leads 
to the production of 8 to 9 ng of bacteria per 
/onole of substrate. It is therefore inferred that 
one ATP per mole is produced during these fer- 
mentations. This is greater by a factor of two 
than is anticipated from the established route of 
butyric acid-acetic acid fermentation and sug- 
gests that additional phosphate esterification may 
be available from anaerobic electron transfer re- 
actions. 

Reduction Deamination op Gltcine. It has 
been found that exhaustive dialysis of partially 
purified enzyme preparations of C. sticklandii 
results in the loss of ability to catalyze the reduc- 
tion of glycine to acetate and the concomitant 
synthesis of ATP. The activity of such prepara- 
tions can be restored by the addition of a heat 
stable factor obtained from the partially purified 
enzyme. The properties of this material are un- 
der investigation. 

One Carbon Metabolism 

In studies with dried cells of Methanobacterium 
omelianski which catalyze the reduction of carbon 
dioxide to methane, it could be demonstrated, in 
collaboration with A. Lezius and F. Lynen, Max- 
Planck Institute, Munich, Germany, that carbon 
monoxide, formaldehyde, formate or methanol are 
not intermediates. C0 2 reduction in the presence 
of molecular hydrogen follows a long lag period 
during which time an extremely oxygen sensitive 
compound is produced. Both C0 2 and H 2 are 
required for the formation of this substance 
which, after reaching an optimal concentration, 



allows methane formation to proceed at a linear 
rate for hours or until the substrates are ex- 
hausted. This lag period is not associated with 
a deficiency in the hydrogenase system nor is it 
overcome by the addition of ATP or ATP-gener- 
ating systems. Once the oxygen labile compound 
is formed, it can be kept for hours in an atmos- 
phere of pure H 2 . 

The overall reduction of C0 2 to methane is 
inhibited by a number of metabolic poisons, no- 
tably by arsenate, iodoacetate, arsenite and low 
levels (1(H to KHM), 2,4-dinitrophenol. Inhibi- 
tion by the latter compound suggests that coupled 
electron transport phosphorylation occurs during 
C0 2 reduction to methane. The fixation of C 14 2 
into the carboxyl groups of pyruvate and alanine 
by cell-free enzyme preparations was found also 
to be inhibited by dinitrophenol. 

Coenzyme and vitamin analyses revealed that 
two different strains of methane producing bac- 
teria contain exceptionally high concentrations of 
biotin and vitamin Bi 2 coenzyme. The extraordi- 
narily high level of these vitamins in organisms 
whose energy metabolism is restricted to the re- 
duction of C0 2 to methane, is inferential evidence 
that they are catalysts in this biological process. 

Lipoate in Metabolism of Lactate 

Previous studies by Barker and coworkers have 
shown that in Butyribacterium rettgeri lipoate 
is required for the fermentation of lactate but 
not of pyruvate. It is thus suggested that in this 
organism lipoate is required for a process other 
than in the oxidation of an a-keto acid. The con- 
clusion is supported by results of the present 
study showing that lipoate is apparently involved 
in the conversion of lactate to pyruvate but not 
in the subsequent dissimilation of pyruvate. The 
oxidation of lactate to acetate and C0 2 by washed 
cell suspensions of B. rettgeri is obligately cou- 
pled with the stoichiometric reduction of oxidized 
lipoate to the reduced form. On the other hand, 
lipoate cannot serve as an electron acceptor for 
the oxidation of pyruvate. 

It has been further established that the fermen- 
tation of pyruvate by cells grown on lactate (in 
the presence of lipoate) are quite different. With 
glucose adapted cells, pyruvate is dismutated to 
a mixture of lactate, acetate and C0 2 whereas 
with lactate adapted cells, pyruvate is fermented 
to butyrate, acetate and C0 2 . 



NATIONAL HEAKT INSTITUTE 



79 



Pyruvic Kinase Action 

A continuing problem is represented by studies 
of the pyruvic kinase mechanism, particularly 
from the point of view of reconciling the abili- 
ties of the enzyme to phosphorylate alternatively 
an enolic oxygen, or inorganic fluoride in the 
presence of bicarbonate. Perhaps the narrowest 
way in which these dissimilar reactions could be 
reconciled would be to place F - on the enzyme 
surface in place of C = C-C~, and C0 3 = in place 
of -CO2 - , and further to propose an intermediary 
acyl phosphate in both reactions. Accordingly 
experiments were carried out to test this hy- 
pothesis, by incubating either pyruvate or phos- 
phoenolypyruvate with crystalline pyruvic kinase 
in H s 2 0, in absence of nucleotides. The enzyme 
was found to catalyze a slow exchange of solvent 
hydrogen into pyruvate, but none into phospho- 
enolpyruvate. This result constitutes evidence 
against the hypothesis. 

Biochemistry of the Differentiating Slime Mold 

Carbohydrate Metabolism. Apparent changes 
in the accumulation of glucose-6-phosphate de- 
hydrogenase during differentiation of the slime 
mold Dictyostelium discoideum prompted an in- 
vestigation, in collaboration with B. Bloom of 
NTAMD, to ascertain if the routes of carbohy- 
drate metabolism undergo significant changes 
also. The relative yields of C 14 2 from 6-C 14 - 
glucose and l-C 14 -glucose were determined when 
these substances were incubated with the slime 
mold at 5 different stages of development. The 
ratio of C0 2 derived from the 6 carbon and the 
1 carbon atom was 0.7 in the amoebae, 1.3 at 
pseudoplasmodium and preclumination stages and 
0.3 at the fruit stages, thus indicating marked 
changes in the relative contribution of the hexose 
monophosphate-pentose-phosphate pathway and 
and glutamic-pyruvic transaminase were not at- 
velopment. Three possible pathways to account 
for the ratio of 1.3 observed at preculmination 
were dismissed as unlikely because of the inability 
to detect the required enzymatic steps in cell- 
free extracts. 

ISOCITRATE AND GLTJCOSE-6-P-DEHYDROGE:NrASES. A 

heat-labile proteolytic-type agent (s) present only 
in extracts prepared at the amoebae stage of de- 



velopment was discovered to destroy specifically 
isocitric- and G6P-dehydrogenases. These en- 
zymes could be fully protected by the presence 
of their substrates. Glutamic acid dehydrogenase 
and glutamicpyruvic transaminase were not at- 
tacked. Knowledge of this differential enzyme 
destruction as a function of the stage of differ- 
entiation has, in conjunction with related studies, 
suggested the possibility that enzyme activities 
in vivo may be controlled by sequential endo- 
genous substrate utilization (and inhibition), 
thus accounting for certain metabolic changes 
occurring during differentiation. 

Enzyme Induction 

The accumulation of a specific enzyme upon 
induction could be due in whole or in part to a 
decreased rate of destruction, as well as to an 
increased rate of synthesis. In other words, a 
"basal level" inducible enzyme may be turning 
over more rapidly than other cellular proteins, 
and the inducer may act by combining with this 
enzyme and stabilizing it, thus allowing its ac- 
cumulation. In an effort to detect possible dif- 
ferences in the turnover of inducible enzymes 
under conditions of high and low rates of syn- 
thesis, a 30 second pulse of highly tritiated leu- 
cine was given to exponentially growing E. coli 
cells in the presence, (a) of melibiose, a power- 
ful inducer of ^-galactosidase, and (b) of galac- 
tose, a poor inducer. The /?-galactosidase formed 
in the presence of melibiose was y s as radioactive 
as the average cellular protein whereas /?-galac- 
tosidase formed in the presence of galactose was 
twice as radioactive as the average cellular pro- 
tein. These results would be expected if in- 
creased enzyme accumulation on induction was in 
part due to a decreased turnover of /?-galacto- 
sidase. 



LABORATORY OF CHEMISTRY 
OF NATURAL PRODUCTS 

The work of the Laboratory may be summar- 
ized in three areas. These are (1) the develop- 
ment and application of new methodology for 
the study of lipids and related substances, (2) in- 
vestigations of the occurrence, structure and prop- 
erties of plant alkaloids, and (3) studies of com- 
ponents of the kallikrein-kallidinogen-kallidin 



80 



ANNUAL, REVIEW OF INTRAMURAL RESEARCH 



system, and of the chemistry of human polypep- 
tide vasodilators. 



Lipid Methodology 

Investigations of lipid metabolic problems im- 
portant to atherosclerosis studies have been ham- 
pered for some time by the poor state of method- 
ology in the field. Work on all aspects of lipids, 
from basic chemistry to nutrition, advanced very 
slowly in the past because of this situation. Some 
time ago this laboratory started work on the de- 
velopment of new laboratory techniques for 
studying naturally occurring lipids and related 
substances, with a view to developing new meth- 
odology. Significant basic discoveries in gas 
chromatography had been made earlier in Eng- 
land, and these were taken as the starting point. 
During 1959 methods were developed for the 
study of fatty acids and related long-chain com- 
pounds including alcohols, aldehydes and bases 
of the sphingosine type. This work required the 
development of new column materials for gas 
chromatographic work, and these column pack- 
ings are now in rather general use in this coun- 
try and abroad in both biological research work 
and manufacturing control laboratories. 

The success of these methods in providing a 
new and important tool for studying long-chain 
compounds obscured the fact that gas chroma- 
tography was in fact more than a specialized 
tool or a new body of techniques of limited use. 
Early experiments suggested that steroids and 
other large molecules were not amenable to sep- 
aration in this way but these experiments did 
not distinguish sufficiently between theoretical 
considerations and problems of technology. Dur- 
ing i960 the problem of extending gas chroma- 
tographic methods to the separation of steroids 
and other complex compounds of natural origin 
was undertaken. A series of studies was made 
with thermostable phases not previously investi- 
gated. The requirements for supports, for de- 
tection systems, for chromatographic columns and 
for vaporizing systems were investigated. Pre- 
vious studies with long-chain compounds pro- 
vided the necessary background of experience. 
It was found that with new techniques many 
classes of high molecular weight compounds could 
be separated. Steroids, alkaloids, vitamins A, D, 
E and K, many drugs and drug metabolites, and 



many metabolic products of varied structure can 
now be separated and studied by gas chroma- 
tography. The sensitivity of these procedures, the 
superb resolving power, and the ability to use 
complex mixtures of a few micrograms or less in 
total size (the ultimate sensitivity of current de- 
tection systems has never been used) are now 
well recognized. 

Alkaloid Work 

Studies of alkaloids of the Amaryllidaceae have 
been continued. Several biosynthetic studi«s 
were started in collaboration with Dr. A. R. Bat- 
tersby. The biosynthetic work is being carried 
out primarily in England, in continuation of Dr. 
Battersby's present studies, and the chemical de- 
gradative work will be done here. Current ex- 
periments are designed to establish the origin of 
the ring systems of these alkaloids. 

Structural studies were also continued. The 
structure of buphanamine was established, and 
additional degradative methods for studying the 
alkaloids were developed. 

Additional supplies of galanthamine were iso- 
lated for physiological investigations. This 
Amaryllis alkaloid was introduced into therapy 
in Russia in 1958 in the treatment of myasthenia 
gravis. Several synthetic compounds, derived 
from the alkaloid, have been prepared for com- 
parisons of activity. 

Studies of Cassia, and Ormosia alkaloids re- 
sulted in the determination of structures for ja- 
maidine and jamaicensine. These compounds are 
found in seeds of Ormosia, jamaieensis; material 
of Jamaican origin was used in the work. The 
relationship of the two alkaloids was demon- 
strated by the synthesis of jamaidine from ja- 
maicensine. The Cassia alkaloid cassine is still 
under study. 

The fungal antibiotic pleurotin was isolated 
from Pleurotus griseus. Structural studies are 
in progress. 

Gas chromatographic techniques useful for 
steroid work were found to be immediately ap- 
plicable to studies of complex mixtures of alka- 
loids. A preliminary survey indicated that many 
groups of alkaloids with widely different ring 
systems and different functional groups could be 
separated. An intensive study of two croups was 
undertaken. The gas chromatographic behavior 



NATIONAL HEART INSTITUTE 



81 



of the Amaryllis alkaloids was investigated, since 
many of these compounds were available from 
earlier studies. The separations on a non-polar 
phase (SE-30) were found to be excellent, and 
it was found that individual alkaloids of the 
Amaryllis group may be characterized in this 
way. 

Morphine alkaloids were also investigated. It 
was found that the complex mixture derived from 
the plant (crude opium) could be extracted and 
the composition of the extract determined by 
gas chromatography. A "fingerprint" of the 
mixture was obtained. This work was carried 
out with a non-polar phase (SE-30). These re- 
sults suggest that gas chromatography will be- 
come a valuable tool in alkaloid work, both in 
isolation and structure proof studies. 

Kallikrein-Kallidinogen-Kallidin System 

The human vasodilating substance kallikrein 
owes its action to the formation of the physiolo- 
gically active polypeptide kallidin. The latter 
compound is formed when kallikrein acts on kal- 
lidinogen, a component of human plasma. Work 
directed to the isolation of kallikrein, kallidin 
and kallidinogen has been carried on as a joint 
study with the Laboratory of Cardiovascular 
Physiology. 

A kallidin fraction was prepared by the action 
of human urinary kallikrein on human plasma 
kallidinogen. Two kallidins resulted when this 
material was purified. These compounds, kalli- 
dins I and II, are similar but not identical poly- 
peptides, and both have vasodilator activity. Cur- 
rent work is directed to the preparation of an 
additional supply of the polypeptides. Prelim- 
inary comparisons have been made with brady- 
kinin, and structural work on the composition 
of the kallidins has been started. 

Studies of human plasma, pancreatic and uri- 
nary kallikrein were continued. Partially puri- 
fied fractions were obtained, and additional work 
is projected to obtain material of greater purity. 
These kallikreins are not identical, but presum- 
ably each one yields the same kallidin. 

Procedures for isolating human plasma kalli- 
dinogen are under development. This compon- 
ent of the system is needed to complete the pro- 
posed studies of kallidin formation. 



LABORATORY OF CHEMICAL 
PHARMACOLOGY 

Development of New Drugs 

Eeserpine Analogues 

It is difficult to maintain a constant clinical 
effect with reserpine due to its cumulative effect 
and its instability in the gastrointestinal tract. 
In collaboration with Ciba Laboratories, reser- 
pine analogues have been developed in which the 
unstable trimethoxy group is replaced by a 
methyl group. This ether exists in two epimeric 
forms (Su 8842 and Su 9064), both of which are 
stable in the gastrointestinal tract and are com- 
pletely absorbed. Furthermore, they act re- 
versibly, sedation and effects on brain 5HT last- 
ing only about 8 hours in rabbits. In man the 
drugs act rapidly and the effects are easily con- 
trolled. Preliminary trials in treatment of psy- 
choses look promising. Furthermore, since low 
doses of drugs cause a persistent depletion of 
peripheral norepinephrine (NE) without releas- 
ing brain 5-hydroxytryptamine (5HT) or evok- 
ing sedation, they will also be tested in the treat- 
ment of hypertension. 

Benzoquinolisines With Reserpinelike Action 

Benzoquinolizines, like tetrabenazine, act 
quickly, producing reserpinelike effects, yet they 
release only small amounts of brain 5HT. This 
suggests that they act directly, perhaps by mi- 
micking 5HT in brain, rather than through re- 
lease of 5HT. A number of sedative benzo- 
quinolizines which do not release brain 5HT are 
under investigation to see whether they exert 
typical reserpinelike effects. 

Imipramine Metabolite 

A metabolite of imipramine, monomethyl nori- 
mipramine, has been found to be about 25 times 
as active as the parent drug in blocking reser- 
pine action. This important lead may provide 
a highly active compound for treatment of men- 
tal depression. 

Drugs for Arthritis and Gout 

A few years ago, these studies were initiated 
when it was found that the two metabolites of 
phenylbutazone isolated in this laboratory were 



82 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



active in man. Metabolite I exerted a pro- 
nounced antirheumatic effect; Metabolite II was 
strongly uricosuric. Now that Geigy Pharma- 
ceuticals Co. has overcome certain synthetic diffi- 
culties, Metabolite I, oxyphenbutazone, has been 
introduced in Europe (Tenderil) as a potent, less 
toxic, antirheumatic agent and it will soon be in- 
troduced in the United States. Metabolite II led 
to development of an analogue, sulfinpyrazone 
(Anturan), now available as an extremely potent 
uricosuric agent for treatment of chronic gout. 

Except for further trials with a methylsulfone 
analogue, which has an extraordinary half -life, 
this project is terminated. 



Problems of Drug Administration in 
Long-term Therapy 

Long-term therapy creates problems in clinical 
toxicity, not easy to foresee from usual animal 
toxicity experiments. 

Biochemically Irreversible Drugs 

Reserpine and monoamine oxidase (MAO) in- 
hibitors are drugs with actions lasting long after 
cessation of dosage despite their rapid biotrans- 
formation. Since the drug effects are irreversible, 
they are usually given in small doses over a 
period of weeks to reach the required effect; 
large priming doses can produce profound toxic 
manifestations in susceptible individuals. This 
poses problems of dosage regulation since the 
effects of these inherently dangerous drugs are 
related not to plasma levels but to the extent to 
which they inhibit biochemical processes. Even 
with small doses drugs with cumulative effects 
can produce a delayed toxic action. For example, 
when Catron (JB 516) was given to dogs in 0.5 
mg/kg doses daily it produced lesions in the in- 
ferior olivary nucleus or in the pyriform cortex 
after 100 days of treatment. 

Effects of Drugs on Endocrines 

Tranquilizing agents (reserpine, chlorproma- 
zine) cause hypersecretion of ACTH and per- 
haps other pituitary hormones when given in 
sedative doses. If these compounds, as is likely, 
stimulate ACTH secretion in man, the biochem- 
ical and physiological consequences of this action 
after long-term treatment become important. 



Drug Combinations 

The use of drug combinations in long-term 
therapy has potential difficulties. A number of 
drugs, MAO inhibitors for example, may depress 
activity of the liver enzymes that inactivate other 
drugs. This potential hazard should always be 
tested especially if the other drug is a central 
nervous system stimulant or depressant. 

One of the two drugs may induce an increased 
activity of the enzyme that metabolizes the other. 
As an example, in using the drug combination, 
phenylbutazone and aminopyrine, phenylbutazone 
induces an increase in the metabolism of amino- 
pyrine so that the latter drug remains in the 
body for a very short time. Similarly, barbitur- 
ates may speed up the metabolism of anticoagu- 
lants and throw control of the coagulation mecha- 
nism out of balance. 

If both drugs act centrally on amine receptor 
sites, a combination of drugs can exert effects 
that neither one causes alone. It has been shown 
that reserpine given to animals pretreated with 
a MAO inhibitor produces profound excitation 
rather than sedation. Perhaps only the fact that 
relatively small doses of reserpine are given to 
man has prevented catastrophic effects with this 
combination of drugs. It has also been shown 
that animals given an MAO inhibitor followed 
by imipramine (both in non-toxic doses) develop 
bizarre symptoms and hyperpyrexia. In patients 
this combination of drugs has caused peculiar 
motor signs, circulatory collapse and hyperpy- 
rexia. Finally, the activity of chlorpromazine 
appears to be markedly increased after pretreat- 
ment with a MAO inhibitor. This has also been 
reported in man. 

Biogenic Amines 

Serotonin (5HT) and Norepinephrine {NE) 
in Brain 

Using the compound a-methyl-m-tyrosine 
(MMT) it has been possible to show that reser- 
pine action is associated with the effects on brain 
5HT rather than on brain NE. In rats MMT 
reduces the NE of brain with almost no change 
in brain 5HT ; the animals are not sedated. How- 
ever, if reserpine is now given to these animals, 
5HT is lowered and sedation ensues. 

With the new tool, MMT, it has been possible 



NATIONAL HEART INSTITUTE 



83 



to show that the amines, released from binding 
sites and stabilized by a MAO inhibitor, elicit 
opposite effects in brain. Thus, if a rat is given 
a MAO inhibitor and then reserpine, excitation 
occurs as amines are liberated but stabilized. In 
contrast, if animals are pretreated with MMT, 
which depletes only the NE, and then given the 
combination of the MAO inhibitor and reserpine, 
only 5HT is stabilized in a free form and the ani- 
mals now show sedation. Other experiments, 
which involve administration of MAO inhibitors 
to animals pretreated with reserpine, show that 
free NE at low levels in brain can elicit all the 
effects of large doses of amphetamine. 

Brain Amines in the Newborn 

Levels of brain amines were related to gross 
behavior in rats of various ages. At birth the 
amine levels are very low. The levels increase 
with age and are normal when behavioral pat- 
terns are developed. Newborn guinea pigs which 
have more fully developed behavioral patterns 
have a normal content of brain amines. The 
newborn rat does not lack ability to synthesize 
the amines but is unable to store them in brain. 

Imipramine (Tofranil) 

Imipramine is a clinically effective antidepres- 
sant drug which does not elicit excitation in nor- 
mal animals or man. It is not an MAO inhibitor 
nor does it alter brain amines. The pattern of 
its pharmacologic effects is that of a weak chlor- 
promazine-like compound. Thus none of present 
screening procedures would show up this com- 
pound as an antidepressant. This action is dra- 
matically evident in dogs and rats treated with 
reserpine. The following effects of reserpine 
are prevented by imipramine: depression, miosis, 
diarrhea, active closure of eyelids, hypothermia, 
potentiation of alcohol and barbiturate anesthe- 
sia, increased activity of central parasympathetic 
system. The activity of chlorpromazine is not 
affected by the drug. Preliminary data suggest 
that imipramine might act centrally by blocking 
the effects of free 5HT, but much more evidence 
for this is needed. 

The latency in action of imipramine in animals 
and man has led to tests of one of its metabolites, 
monomethyl-norimipramine. This substance is 
about 25 times more active in blocking reserpine 
action than the parent compound. A dog given 



reserpine in sufficient dosage to produce coma 
and death is not depressed and lives if pretreated 
with 1 mg/kg of metabolite. 

The discovery of the antidepressant effects of 
imipramine suggests that psychotherapeutic 
drugs with new kinds of activity might be dis- 
covered by screening them for the modification 
of the disturbances produced by other drugs. 

Monoamine Oxidase Inhibitors 

It had been previously shown that the antide- 
pressant action of MAO inhibitors in animals was 
associated with the rise in brain NE and not in 
brain 5HT. It has now been established that 
this is also true for the new non-hydrazine in- 
hibitors. As a result of this work, a rise in brain 
NE is regarded as a sine qua non for evidence 
that a compound is a MAO inhibitor antidepres- 
sant. 

In studies of mechanism by which the inhibi- 
tors lower blood pressure, measurement of the 
postganglionic electrical responses induced by 
preganglionic stimulation of sympathetic ganglia 
indicates that the hypotensive effects are not due 
to inhibition of synaptic transmission. 

Hypotensive Drugs, Guanethidine and Bretylium 
Guanethidine has been shown to lower blood 
pressure by depleting NE from peripheral nerve 
endings. Its action is unusually specific for it 
does not release peripheral 5HT nor either amine 
from brain. Bretylium, a quaternary ammonium 
compound, lowers blood pressure by preventing 
the physiological release of NE from nerve end- 
ings. 

Preliminary experiments on the mechanism of 
action of these compounds were based on the con- 
cept suggested by Burns and Rand that post- 
ganglionic adrenergic fibers may actually be 
cholinergic, with acetylcholine at nerve endings 
liberating NE, which in turn acts on muscle as 
a local hormone. Bretylium (at higher doses a 
ganglionic blocker) would then be a specialized 
cholinergic blocking agent with an affinity for 
sympathetic nerve endings and guanethidine (at 
higher doses a ganglionic stimulant) a specialized 
cholinergie stimulant which depletes NE by re- 
leasing acetylcholine at sympathetic nerve end- 
ings. Preliminary experiments favor this view 
since pretreatment of rats with bretylium antago- 
nizes the NE-releasing action of guanethidine. 



84 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



These drugs should prove useful tools in studies 
of the acetylcholine-NE relationship throughout 
the sympathetic nervous system. 

NE in Sympathetic Synaptic Transmission 

The function of the brain amines is difficult to 
study since functional units in the central nervous 
system are difficult to isolate. Since sympathetic 
ganglia contain considerable NE, the effects of 
depleting the amine were studied. After gangli- 
onic NE has been depleted with reserpine, the 
postganglionic response to preganglionic electrical 
stimulation is greatly exaggerated. Similar re- 
sults are obtained by the administration of 
adrenergic blocking agents and by bretylium. In 
contrast, if free NE is pooled in ganglia by the 
administration of an MAO inhibitor followed by 
reserpine, virtually complete ganglionic blockade 
results until the free amine diffuses away. These 
results suggest that the transmission across sym- 
pathetic synapses is regulated both by acetylcho- 
line and NE and that during cholinergic gangli- 
onic stimulation signals are put out to release NE, 
which in turn inhibits the effect of acetylcholine 
on synaptic transmission. This feedback mech- 
anism would serve to prevent wide swings in gan- 
glionic activity which otherwise might occur with 
changes in sympathetic output. Current investi- 
gations are aimed at determining whether NE 
and 5HT in brain also act by modulating synap- 
tic transmission. 

Reserpine Action 

It is important to know the precise pharmacol- 
ogy of reserpine since it may serve to map out 
a neural organization in brain, modulated by 
5HT. It has been proposed that such a neuronal 
system integrates parasympathetic with extrapyr- 
amidal and emotional functions (trophotropic 
system of Hess) and is stimulated by reserpine, 
whereas an opposing adrenergic system (ergo- 
tropic system of Hess) is inhibited by chlorpro- 
mazine. Pharmacologically, reserpine acts oppo- 
sitely to chlorpromazine in that it stimulates 
central parasympathetic activity; chlorpromazine 
in contrast depresses central sympathetic activity. 
These stimulatory effects of reserpine are blocked 
by chloralose; those of chlorpromazine are not. 

Electrophysiological measurements also show 
the difference between the two drugs. Chlorpro- 
mazine has its action on the reticular activating 



system by blocking stimulation from collateral 
input. Reserpine in therapeutic doses has its 
main action on the limbic system, producing a 
spontaneous electrical activity in the amygdala 
which spreads to the rest of the limbic system, 
but not to the neocortex. Chlorpromazine does 
not exert this action in therapeutic dosage. 

Histamine 

Although for many years histamine has been 
recognized as a normally occurring agent with 
marked pharmacologic effects, the question of its 
physiologic role remains unanswered. Projects 
in this laboratory designed to shed new light on 
this question have led to a sensitive and specific 
fluorometric method for the assay of histamine 
and for the enzymes responsible for its metabo- 
lism, diamine oxidase and imidazol-N-methyl 
transferase. It has been established that "his- 
taminase" and diamine oxidase are the same en- 
zyme, but that the diamine oxidases from various 
organs exhibit measurable differences. Experi- 
ments on the release of histamine from rabbit 
platelets have led to the demonstration that 
thrombin is an extremely potent histamine re- 
leasing agent, and that thrombin generation may 
be involved in the physiologic release of hista- 
mine from platelets. 

Studies in Biochemical Behavior 

Studies have been instituted to determine how 
body biochemical reactions are adapted to changes 
in environment through central nervous activity, 
and how these control mechanisms are affected by 
drugs through hypothalamic-pituitary-endocrine 
function and the autonomic system. 

(1) In rats, cold, alcohol and depot ACTH 
elicit the same rise in plasma corticosterone, 
plasma free fatty acids, and liver tryptophan- 
peroxidase indicating that cold and alcohol re- 
lease ACTH. Depot ACTH, however, produces 
a much greater rise in liver triglycerides, suggest- 
ing that this hormone can also exert an effect on 
lipid metabolism not mediated through the adre- 
nals. 

(2) Reserpine and allied compounds produce 
hypersecretion of ACTH. They act only in doses 
that deplete brain 5HT by 50 percent, a dose also 
required to evoke sedation. The response is not 
a "non-specific stress" since isoreserpine, which is 



NATIONAL HEART INSTITUTE 



85 



non-sedative, does not stimulate the pituitary even 
in lethal doses. Keserpine in doses of 2 mg/kg 
depletes pituitary ACTH by 80 percent over a 
period of 20 hours. Thus published reports that 
reserpine can prevent responses to stress are ex- 
plained by the depleted pituitary. This was 
shown by experiments in which the pituitary 
ACTH of animals, cold-exposed for 20 hours, was 
also found to be drastically lowered, and the ani- 
mals could not respond to a further stress. 

(3) Chlorpromazine and other phenothiazines, 
but only in sedative doses, also cause the pituitary 
to release ACTH. This is not a "nonspecific" 
stress since non-sedative phenothiazines do not 
stimulate the pituitary even after huge doses. 
Central stimulants like amphetamine stimulate 
the pituitary only transiently. Thus the tran- 
quilizing drugs produce an effect on the pituitary 
usually associated with stress. 

(4) Studies suggest that the autonomic nervous 
system as well as the pituitary is involved in 
drug-induced deposition of triglycerides in liver. 
Thus ganglionic blocking agents prevent both the 
alcohol-induced triglyceride deposition in liver 
and the mobilization of fatty acids from adipose 
tissue without affecting ACTH hypersecretion. 

(5) More than one mechanism is involved in 
aberrations of lipid metabolism induced by drugs. 
Carbon tetrachloride, but not alcohol, for exam- 
ple, produces deposition of triglycerides in the 
liver of hypophysectomized rats. Drug induced 
changes in lipid metabolism are effected by at 
least three mechanisms: (a) increased mobiliza- 
tion of free fatty acids from adipose tissue (alco- 
hol, CC1 4 and ethionine), (b) activation of tri- 
glyceride synthesis in liver (alcohol), and (c) 
blocking of triglyceride removal from liver 
(CCI4). 

Passage of Substances Across Membranes 

Membranes Within the CNS 

It has been shown that if various sugars such 
as insulin, sucrose and mannitol are injected into 
the lateral ventricle they enter the brain through 
the ependymal lining slightly if at all. However, 
within 1 hour they have passed completely into 
the cisterna magna. Although these substances 
cannot enter the cerebrospinal fluid from plasma, 
they do pass from CSF into plasma rapidly and 



all at the same rate. These results indicate that 
they leave by fluid flow through large pores, prob- 
ably in the arachnoid villi. It is possible that the 
rate at which the large molecules leave is related 
to rate of CSF turnover. 

Penetration of Drugs into Cells 

As reported previously, basic organic com- 
pounds cross the red cell at rates related to lipid- 
solubility. Organic acids also penetrate the red 
cell at rates roughly related to lipid solubility, 
but in addition completely ionized compounds 
like phenol red readily penetrate. Organic anions 
probably enter red cells by diffusing through 
large positively charged pores, the mechanism 
perhaps resembling that involved in the entry 
of chloride. 

Active Transport Mechanisms 

Data obtained in studies of the passage of 
pyrimidines across everted intestinal sacs (rat) 
suggest that there must be an OH group in the 
6-position of the pyrimidine or purine ring for 
a compound to interact with the pyrimidine trans- 
port mechanism, either as participant or inhibitor. 
The nature of substituents in the 2 or 5 position 
seems unimportant. Two antitumor agents, 5- 
fluorouracil and 5-bromouracil, are actively trans- 
ported across the intestinal epithelium by the 
pyrimidine mechanism. These are the first ex- 
amples of drugs that are absorbed by active 
transport. Although purines (xanthine, uric 
acid, etc.) block pyrimidine (uracil) transport, 
they are not themselves transported. 

The transport of pyrimidines is an adaptive 
process. Starvation of rats, for 4 days, increases 
the active transport of uracil by about 600 per- 
cent without the changing passive component. 
Hypophysectomy, but not adrenalectomy, also in- 
creases active transport by 250 percent. It is 
possible that lowered food intake is the common 
factor in starvation and hypophysectomy. 

Transport of Catecholamines 

The previously reported finding that 5HT is 
taken up in platelets by active transport has led 
to experiments designed to ascertain whether neu- 
rohormones in general and catecholamines in 
particular are also held in neurons by a "pump" 
mechanism. An important finding has been that 
labelled NE is also taken up by tissue slices by 



86 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



a mechanism which obeys all the criteria of active 
transport. This process is blocked by cardiac 
glycosides and by reserpine. The process is pres- 
ent in brain, heart, kidney and pineal gland and 
absent from liver and muscle. Calculation of 
uptake of labeled amine indicates that the endog- 
enous NE is present in tissue in two pools, only 
one of which is readily miscible with exogenous 
amine. This fits a picture of "free" amine in 
neurons maintained by the pump mechanism, and 
amine in granules bound to some cellular con- 
stituent. 

A number of drugs inhibit uptake of NE in 
vitro, perhaps by competing for the transport 
process. These consist of sympathomimetic (co- 
caine and amphetamine) and sympatholytic 
(chlorpromazine) agents, depleters of tissue cate- 
cholamines (guanethidine) and thyroxine. Most 
of these compounds are known to enhance the 
action of administered catecholamines. This sug- 
gests that the uptake mechanism is an important 
means of terminating biological action of cate- 
cholamines. 

Effect of Ouabain on Phosphatidic Acid 

The synthesis of phosphatidic acid in rabbit 
brain slices is inhibited by ouabain in a concen- 
tration as low as 10 -7 M and is 50 percent inhib- 
ited at 10 -5 to ICh 8 M. Higher concentrations of 
ouabain do not inhibit the synthesis of phospha- 
tidic acid any further. 

Drug Metabolism 

Antimetabolites 

6-Chloropurine (6-C1P) and its metabolite, 
6-chlorouric acid, markedly inhibit the xanthine- 
oxidase uricase pathway. Both xanthine oxidase 
and uricase are blocked in vitro at concentrations 
corresponding to tissue levels in vivo. 6-C1P 
inhibits synthesis of RNA and DNA only slightly 
but markedly inhibits total lipid synthesis in liver 
slices. 

6-Methylaminopyrine (methylated adenine or 
MAP) is a purine of unknown function occurring 
in trace amounts in cytoplasmic RNA. A methyl - 
ating enzyme in rat liver homogenate transfers a 
methyl group from labeled methionine to form 
MAP mainly in cytoplasm. 



Microsomal Drug Oxidation 

This mechanism continues to command atten- 
tion since it involves direct utilization of oxygen 
from air; it may share the same active oxygen 
donor as cholesterol; and a similar mechanism 
may hydroxylate steroids to form corticoids. In 
the last report it was proposed that TPNH + 2 
react with a constituent of microsomes to form a 
hydroxyl-donor which oxidizes substrate, and in 
absence of substrate forms H 2 2 . However, 
kinetic studies now show that the H 2 2 cannot 
be formed from the hypothetical hydroxyl donor. 

In view of the fact that cholesterol is formed 
in microsomes by a TPNH-requiring enzyme it 
may be of some importance with respect to mech- 
anism that triparanol (MER 29) used clinically 
to block cholesterol synthesis also inhibits the 
metabolism of many drugs in vitro. 

Induced Enzyme Formation 

The mechanism by which many drugs increase 
the ability of rats to metabolize the same or 
closely related drugs is being actively pursued. 
The increased enzyme activity produced by pre- 
treatment of rats with phenobarbital, phenyl- 
butazone, chlorcylizine (an antihistaminic) and 
aminopyrine, is paralleled by accelerated drug 
metabolism in the intact animal and by a shorter 
duration of drug action. In man phenylbutazone 
also accelerates the metabolism of aminopyrine, 
and barbiturates seem to enhance the metabolism 
of certain anticoagulants. In rats the inductive 
effects are not prevented by starvation, by adren- 
ergic blocking agents, or by lowered thyroid ac- 
tivity. 

Studies with Ascorbic Acid 

Many drugs not only induce the activity of 
drug enzymes in liver microsomes but stimulate 
synthesis of L-ascorbic acid by mechanisms that 
do not involve the pituitary-adrenal system. In 
studies of this mechanism of increased ascorbic 
acid synthesis it was found that UDPG dehydro- 
genase, a key enzyme in the series of reactions: 

glucose >- uridinediphosphoglucose 

UDPG dehydrogenase uridinediphosphoglucu- 
Je- 
rome acid >- D-glucuronic acid-l-PO*4 y 

D-glucuronic acid >- L-gulonic >- 

L-ascorbic is stimulated by methylcholanthrene. 



NATIONAL HEART INSTITUTE 



87 



In addition, this hydrocarbon increased activity 
of enzymes which metabolize ascorbic acid. The 
possibility has been mentioned in other reports 
that ascorbic acid may have a role in drug metab- 
olism. In accord with this view the muscle re- 
laxant action of Flexin is unusually prolonged 
in ascorbic deficient guinea pigs due to decreased 
activity of the Flexin-metabolizing enzyme in 
liver microsomes. However, other factors such 
as lowered protein intake might also explain this 
effect. 

Of unusual biochemical interest is the finding 
that the 5-carbon analogue of ascorbic acid, L-ery- 
throascorbate, can be formed from ascorbic acid 
in rat liver (decarboxylation of ascorbic to L- 
xylonic acid, followed by conversion to the ascor- 
bic analog). The role and function of this 
substance might be of considerable interest. 

Some of the symptoms of scurvy in guinea pigs 
are reversed by 3-methylcholanthrene by an un- 
known mechanism. These include a marked re- 
duction in incidence of hemorrhage, reduction of 
bone resorption, preservation of dentinogenesis 
and prevention of death of odontoblasts. 

Chemical Inhibition of Cholesterol Synthesis 

Because of the possibility that compounds 
which inhibit conversion of mevalonic acid to 
cholesterol might not affect other metabolic proc- 
esses, the search for antagonists has continued. 
Three analogues of mevalonic acid, which sup- 
press the incorporation of mevalonic-2-C 14 into 
cholesterol in rat liver homogenates, have the 
following order of potency : 3-hydroxy-3-methyl- 
valeric acid, A 2 -3-methylpentenoic acid, A 3 -3- 
methylpentonoic acid. These antagonists are not 
particularly potent. 



Development of Methods of Analysis 

(1) A method for estimation of imipramine 
and its metabolite monomethyl-norimipramine 
has been developed. A method which separates 
these two substances is important since the action 
of imipramine is probably mediated through the 
highly potent metabolite. 

(2) A method for chlorpromazine which in- 
volves oxidation to the highly fluorescent sulfox- 
ide. This should be applicable to humans in 



therapeutic dosage and may answer some of the 
vexing problems with this drug. 

(3) A method for direct determination of tri- 
glycerides in liver. 

(4) A new and much more rapid method for 
determination of ascorbic acid in tissues. 

(5) A method has been developed for meas- 
urement of cotinine in tissues. Since this is a 
metabolite of nicotine, and is fairly stable in ani- 
mals, it might provide a good measure of the 
amount of nicotine inhaled from cigarettes. 

(6) A method for the tissue estimation of 
Librium, a widely used tranquilizer, has been 
developed. 



LABORATORY OF TECHNICAL 
DEVELOPMENT 

The further development and application of 
gas chromatographic methods to problems of bio- 
chemical and medical research are continuing. 
Additional information has been acquired on the 
range and dependability of combined mass and 
radioactivity detection systems. This has been 
applied to the solution of specific problems in 
collaborative research projects with scientists 
from other laboratories and institutes. 

Efforts have been continued to develop the 
techniques for measuring the quantity and radio- 
activity of labeled materials utilizing the anthra- 
cene column scintillation method previously de- 
vised in this laboratory combined with various 
mass detectors. Its utility in the study of sterols, 
a situation in which high temperatures reduced 
the efficiency of the original method, has been 
extended; the modified method provides a modi- 
fied collector that can be cooled by drawing 
through it cool air in addition to the column 
effluent. 

Leakage of radioactivity into the gas stream 
and penetrating radiation were shown to make 
the argon ionization detector unsuitable for low 
level radioactivity assay. A non-radioactive, non- 
destructive, high-sensitivity detector was required. 
Special electronic circuits were developed for this 
purpose. 

Application of gas chromatographic methods 
to a wide variety of biological problems has 
shown that no single detector is universally satis- 
factory. Requirements for sample recovery or 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



for trapping radioactivity in an anthracene col- 
umn preclude the use of highly destructive elec- 
tric discharges or radioactive ion sources. Radio- 
activity detection on anthracene columns must be 
checked for the presence of quenching materials 
in the effluent. Some ionization systems require 
a very low level of organic vapors to operate 
efficiently and in the case of fluorinated esters of 
the amino acids, the halogen quenched the ioniza- 
tion so that the RF detector proved more suitable. 

These applications have indicated that the most 
effective use of gas chromatography requires at- 
tention to a great many details of physical condi- 
tions, as well as consideration of chemical prop- 
erties. A particular example of simplification by 
control of conditions was the idea of adding 
ammonia to the carrier gas to make possible the 
chromatography of the amino acid esters without 
N-substitution. 

In the course of exploring methods for using 
an ion chamber as a radioactivity detector, it was 
noted that unlabeled organic vapor flowing 
through an unpolarized ion chamber produced 
currents the sign and magnitude of which were 
different for different vapors and easily measured 
by the vibrating reed electrometer. This may be 
a serious obstacle to application of the ion cham- 
ber, but the effect may be applicable as another 
type of detector for gas chromatography or other 
applications. 

The study of detectors has been extended to 
the examination of the applicability of the hydro- 
gen flame, as well as other detectors, to liquid- 
liquid chromatography effluents. Preliminary 
experiments have shown that it is possible to 
volatilize the solvent and convey the less volatile 
solute into the hydrogen flame to utilize the high 
sensitivity of this device for indicating the pres- 
ence and quantity of a component coming off a 
column. 

The collaborative research projects carried on 
with other scientists have made possible the anal- 
ysis of all but a few amino acids, tryptophan 
being especially resistant and with arginine and 
tyrosine yielding multiple derivatives. 

A study on the relationship of the composition 
of serum lipoproteins to the lipids of the dietary 
intake has been carried out in collaboration with 
Dr. Kayden at New York University. The re- 
sults indicate that lipoprotein composition reflects 
the dietary fat composition for at least a day or 



two, and indicate the need for rigid dietary con- 
trol in studies of the circulating lipids. 

Several other collaborative projects were car- 
ried on as joint efforts and members of this labo- 
ratory have participated in the design of the 
experiments as well as in the adaptation of gas 
chromatographic methods to the specific problems. 

The ultrasonic velocity gas chromatography 
detector developed in this laboratory has been 
improved by redesign of the cell. Its perform- 
ance has been shown to conform with theory. It 
is unique in that, without destroying the sample, 
it provides high sensitivity, and very rapid, pre- 
dictable response. 

The application of the electronic frequency re- 
sponse correcting circuit for use with intravascu- 
lar catheters has been improved by simplification 
of the procedure by circuit stabilization and de- 
velopment of a square wave test procedure for 
matching the circuit to the specific catheter. 

Analogue devices for analysis of biological 
problems have been set up in response to specific 
problems presented by scientists from other labo- 
ratories. A method of simulating regurgitation 
curves partially accomplished last year is now 
complete. Fourier series, sums of exponential 
functions and distribution curves are being ana- 
lyzed with equipment developed in this labora- 
tory. 

The development of a general theory of trans- 
port phenomena in linear biological systems has 
been continued and applied to the analysis of 
experimental data on fatty acid metabolism. The 
theory is being developed to provide a formal 
language and method for analysis of a large 
number of biological problems. 

The catheter tip ascorbic acid detector has 
been perfected. The method now has practical 
utility in observation of the form of indicator 
dilution curves for the detection of cardiac shunts 
and determination of the extent of heart valve 
regurgitation. Special waveforms, electrode de- 
signs and techniques of application are under con- 
tinued study to eliminate remaining artifacts 
which stand in the way of quantitative determi- 
nations of flow by indicator dilution curves. 

In a cooperative project with the Surgery 
Branch, a study was undertaken to determine the 
physico-chemical properties of plastics responsi- 
ble for initiation of clotting or unfavorable tissue 
response. The contract services of Battelle Me- 



NATIONAL HEART INSTITUTE 



89 



morial Institute were engaged to provide pure 
specimens for in vivo testing at NIH; physical- 
chemical characteristics were determined at Bat- 
telle. While there were several encouraging 
materials in the group tested, no clear pattern of 
properties has yet emerged. This project was an 
exploratory program to initiate the application 
of industrial research technology, through con- 
tract, to a pressing biological problem. A univer- 
sity group which has shown considerable interest 
and competence in the field was encouraged to 
continue the work. 

A practical method for the rapid analysis of 
micro-milliliter samples of renal tubular fluid is 
still being pursued. Sample handling and alkali 
metal recoveries continue to be inadequate. Sen- 
sitivity and the possibility of a relatively simple 
method encourage continued effort to utilize the 
helium glow discharge to produce the characteris- 
tic alkali metal emission. 

Phosphorescence excitation, emission spectra, 
and emission lifetimes have been determined for 
a series of substituted benzoic acids in acid and 
alkaline media to show the relationship between 
structure and phosphorescence. In general, the 
experimental observations have shown that phos- 
phorescence lifetimes and intensities fall into 
series that relate to the pi electron distribution. 
It would appear, therefore, that phosphorescence 
intensity and lifetime data can be used for char- 
acterization. On the other hand the data obtained 
could be used to predict phosphorescent behavior. 

The application of nuclear magnetic resonance 
to the development of a practical flowmeter has 
progressed in the direction of improved electronic 
methods for the reproducible high sensitivity de- 
tection of the proton signal. Improvement in 
sensitivity makes it possible to show flow effects 
in a 2 mm tube and the detection of trace quan- 
tities of paramagnetic salts. 

The electronic refinements developed in the 
course of the flowmeter application are equally 
applicable to detection of paramagnetic indicators 
and susceptible nuclei for application to, as yet, 
undefined problems. 

LABORATORY OF CARDIOVASCULAR 
PHYSIOLOGY 

The activities of the Laboratory of Cardiovas- 
cular Physiology during 1960 included the ini- 



tiation of several new and interesting lines of 
investigation, but it was predominantly a year hi 
which previously initiated, long range projects 
were brought to fruition. The information gath- 
ered has proven to be meaningful and has per- 
mitted a considerable degree of systematization 
and generalization. 



Homeometric Autoregulation in the Heart. 

One type of intrinsic response exhibited by the 
isolated heart is the well known Frank-Starling 
mechanism. This endows the ventricles with 
performance characteristics such that the heart 
ejects whatever volume is put into it. If inflow 
is augmented and end diastolic pressure and fiber 
length are thus increased, the ventricle contracts 
more forcefully and expels an augmented stroke 
volume. This occurs on a beat-to-beat basis and 
may be designated as heterometric autoregulation. 
This year an intensive study of a second type of 
autoregulation in the isolated heart, one which 
apparently does not utilize the Frank-Starling 
mechanism, was initiated. Homeometric auto- 
regulation, as we have called it, requires at least 
a few beats to develop fully after an increase in 
activity (the tension developed per unit of time). 
The ventricle then exhibits performance charac- 
teristics of such a character that its end diastolic 
pressure and fiber length tend to be maintained 
at or near the initial level because of an increase 
in myocardial contractility. The intrinsic abil- 
ity of the ventricle to rearrange its contractility 
in this fashion is construed to be a matter of sub- 
stantial importance in the understanding of car- 
diac function. Current studies are designed to 
determine whether, when homeometric autoregu- 
lation occurs, a net loss of potassium from the 
heart takes place and whether this can be reason- 
ably construed as being related to the increased 
contractility. 

The Atrium 

Mitral Valve Closure 

Further evidence was acquired to indicate that 
mitral valve closure can be made to occur solely 
as the result of atrial activity. Data were also 
obtained indicating that the contractility of the 
atrium, rather than the level of blood flow, is 



90 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the primary determinant when atrial activity pro- 
duces mitral valve closure. 

Left Atrial and Left Ventricular End Diastolic 
Pressure 

Since, at a constant level of sympathetic ac- 
tivity, the stroke work of the ventricle is a func- 
tion of its end diastolic pressure (fiber length), 
and since the mean atrial pressure constitutes the 
central pressure which must be exceeded by re- 
turning venous blood, the level of mean atrial 
pressure necessary to produce any given ventricu- 
lar end diastolic pressure was submitted to ex- 
amination. It was found that sympathetic stim- 
ulation lowers and parasympathetic stimulation 
substantially elevates mean left atrial pressure 
relative to the left ventricular end diastolic pres- 
sure. The change with parasympathetic stimula- 
tion occurs without any observable effect on ven- 
tricular contractility and is the result of altera- 
tion in atrial function. These studies have been 
extended to man by the Cardiology Section. 

Catechol Amine Metabolism of the Heart 

The observation that cardiac sympathetic nerve 
stimulation regularly produces measurable in- 
creases in the concentration of catechol amine in 
coronary venous blood was substantiated. With 
the use of the trihydroxy indole method, the 
coronary venous output of catechol amine was 
shown to be a function of the stimulation fre- 
quency used. 

It was determined that di-isochloroproteranol, 
which renders the heart unresponsive to cardiac 
sympathetic nerve stimulation, does not do so 
by blocking the release of norepinephrine for the 
latter was readily recovered in large amounts in 
coronary venous blood under such circumstances. 
By implication, therefore, the block must occur 
further down the line. 

It was determined that the initial pressor re- 
sponse to bretylium tosylate, a recently intro- 
duced antihypertensive agent, can be attributed 
at least in part to an initial release of catechol 
amine from the heart, since in the minutes after 
the administration of this agent, increased 
amounts of catechol amine were found in coro- 
nary venous blood. This finding coincided in 
time with an observed positive inotropic and 
chronotropic myocardial effect. 



Peripheral Circulation 

Carotid Sinus Activity and Oxygen Consumption 
Whereas an extremity at rest exhibits a slight 
decrease in its oxygen consumption when carotid 
pressure is lowered, the same intervention will 
substantially increase oxygen consumption in that 
extremity if it is exercising. This important 
finding is in line with the view that a major 
physiologic responsibility of the baroreceptor is 
to safeguard the biochemical status of peripheral 
tissues as their activity varies. The pathways 
(effect on atrial and ventricular function) by 
means of which this is at least partially accom- 
plished is outlined in last year's annual report 
and current publications. 

The effects of direct sympathetic nerve stimu- 
lation indicate that the increased flow in the ex- 
ercising limb results from a functional sympa- 
tholysis, which is a graded curvilinear function 
of the local oxygen consumption. 

Experiments employing the cannulation of 
lymphatics and the collection of lymph from the 
lower extremities of dogs at rest and during elec- 
trically induced exercise have been begun with a 
view to ascertaining whether an increase in kal- 
likrein activity occurs in the perivascular, space 
during exercise to account at least partiaUy for 
the observed functional sympatholysis. This 
study is still in its early stages. 

The Kallikrein System 

Further investigation of the pharmacologically 
active hypotensive proteinases called kallikreins 
have revealed that the kallikreins derived from 
a variety of sources hydrolyze those synthetic 
substrates which contain arginine as the specific 
amino acid residue. The major portion of the 
esterase activity in the urine of normal man, as 
measured by its ability to digest p-toluene-sul- 
fonyl-L-arginine methyl ester, is due to kalli- 
krein. Attempts to differentiate plasma kalli- 
krein from the permeability factor of Miles and 
Wilhelm or the glass-activated proteinase have 
been unsuccessful. On the other hand data have 
been obtained which would suggest that the 
plasma of patients deficient in Hageman factor 
are not deficient in kallikreinogen, but rather 
lacking in kallikreinogenase or some prior enzy- 
matic step required for the activation of plasma 



NATIONAL HEART INSTITUTE 



91 



kallikrein. Partially purified human urinary 
kallikrein has been shown to be antigenic in rab- 
bits and the antibody is capable of inhibiting the 
vasodilator activity of several of the kallikreins. 
Two active polypeptides, called Kallidin I and 
II, have been isolated from human plasma fol- 
lowing the action of human urinary kallikrein. 
Methods have been developed during the year 
which have made it possible to purify human 
urinary kallikrein an additional 20-fold; human 
pancreatic kallikrein, 10-fold ; and human plasma 
kallikrein, 30-fold. 

Reflex Factors in Renal Vascular Resistance 

The diuresis observed during stimulation of 
the stellate ganglion led to a series of studies con- 
cerning the effect of carotid sinus stimulation on 
renal vascular resistance. Although this prob- 
lem has been approached by numerous investi- 
gators, the reported data have seemed to be more 
confusing than enlightening. 

Experiments were designed to determine not 
only whether an increased renal vascular resist- 
ance occurs during carotid hypotension but also 
whether such a resistance increase is exclusively 
intrinsic (autoregulatory) or is, to an appreciable 
extent, attributable to sympathetic stimulation. 
In lightly pentothalized dogs, occlusion of both 
common carotid arteries produced an increase in 
calculated renal vascular resistance as measured 
by clearance techniques. A method was then 
designed by means of which the blood flow 
through one kidney could be directly metered 
without producing any ostensible disturbance of 
the renal innervation. This was accomplished 
by hooking an angled occlusive cannula into the 
lumen of one renal artery after introducing it 
from below through an iliac artery and then per- 
fusing that kidney at a metered, constant flow 
before and during carotid artery occlusion. Dur- 
ing carotid hypotension renal arterial pressure 
rose markedly, the increment sometimes exceed- 
ing that in the aorta. The responses in this type 
of experiment have been consistent; sympathetic 
impulses, reflexly engendered by the carotid sinus, 
produce substantial changes in renal vascular re- 
sistance and blood flow. This study, among other 
things, suggests that the consequences of a change 
in carotid sinus pressure itself can influence blood 
volume. 



Pulsus Alternans 

Alternating End Diastolic Fiber Length as a 
Causative Factor 

The variety of techniques and preparations 
now available makes possible a comprehensive 
and sophisticated analysis of myocardial phe- 
nomena. One example in point is the definitive 
analysis of pulsus alternans. Observations on this 
phenomenon have been made in the dog and cat 
papillary strip, the isovolumetric, innervated in 
situ dog heart and the isolated, supported dog 
heart. The data obtained are not consonant with 
the most popularly held explanations, namely (a) 
a primary alternating impairment of contractility 
from beat to beat; (b) an alternating deletion of 
fractionate contractions, and (c) an alternation 
of the excitation mechanism. The data do indi- 
cate that, when pulsus alternans occurs, the 
weaker beat (WB) can be accounted for on the 
basis of a shorter initial fiber length from which 
the WB initiates. The shorter fiber length prior 
to the WB is due to an inadequate period for 
diastole; this does not allow time for complete 
relaxation and/or a comparable period for filling. 
This limited diastole is due to the large percent- 
age of the total cycle time (larger stroke volume) 
required for systole in the previous, stronger 
beat (SB). The WB has a shorter systolic period 
due to its smaller stroke volume. This allows a 
longer period for relaxation and filling and thus 
a longer initial fiber length to be acquired prior 
to the SB. Thus pulsus alternans occurs when 
myocardial contractility is continuously depressed 
relative to the existing heart rate and stroke vol- 
ume. When, under these circumstances, con- 
tractility is increased (shortening of systole), as 
by norepinephrine, stellate stimulation or homeo- 
metric autoregulation, pulsus alternans abates. 

Chemoreceptors 

The Carotid Body 

This laboratory has made a major research in- 
vestment in the investigation of baroreceptors as 
sense organs which reflexly influence the circu- 
lation. More recently attention has been turned 
to the chemoreceptors. Investigations of the re- 
flex effects upon the heart and systemic circula- 
tion of stimulation of the carotid bodies with 
hypoxic and hypercapnic blood were made in 



92 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the open-chest dog. The carotid sinus-body area 
was independently perfused using a double disc 
oxygenator system which permitted rapid change 
from blood of one gas composition and pH to 
that of a different composition and pH without 
changing perfusion pressure. Continuous meas- 
urements of the pC>2 (polarographic) and pH 
of the perfusing blood were made. Caudal and 
cephalic blood flow were separately measured 
utilizing a Potter electroturbinometer and a Ship- 
ley-Wilson rotameter respectively. Left ventric- 
ular end diastolic pressure, aortic pressure and 
heart rate were simultaneously recorded. Puz- 
zlingly, bradycardia is the primary heart rate 
response to carotid body stimulation confirming 
the findings of deBurgh Daly and Scott. Pre- 
liminary data suggests that this negative chrono- 
tropic effect is due principally to parasympa- 
thetic activation but that, in addition to the 
pathways found by deBurgh Daly and Scott, 
sympathetic withdrawal may also be present. 
These data permitted construction of ventricular 
function curves relating left ventricular end di- 
astolic pressure and stroke work at constant heart 
rate (electrically paced). The relative effects of 
increased peripheral vascular resistance and 
changes in ventricular contractility, and the roles 
of neural and humoral factors in these responses 
are being investigated. 

Rise in Arterial Pressure Following Occlusion 

The rise of arterial pressure following occlu- 
sion of the celiac and mesenteric arteries in the 
cat under chloralose has previously been ascribed, 
on the basis of previous work in this laboratory, 
to a pressoreceptor reflex. It now appears that, 
at least for the time being, this position must be 
revised. The results of a variety of more recently 
conducted experiments which included completely 
isolating the splanchnic vascular bed, both with 
and without cross perfusion, and the recording 
of action potentials, indicate that both mechan- 
ical and reflex factors are operative. The oc- 
clusion of vessels per se increases the peripheral 
resistance. Also, blood runs off from vessels 
distal to the occlusion into the active circulation 
and so tends to increase heart output by acting 
as an infusion. Pressor reflex effects due to local 
irritation as ligatures were tightened on arteries 
were regularly found, but when these were ex- 
cluded in cross perfusion experiments, lesser re- 



flex pressure changes could still be elicited by 
arresting the flow in splanchnic vessels. Whether 
pressoreceptors, chemoreceptors or ischemic pain 
receptors are involved remains to be established. 

Mammalian Myocardium 

Some progress has been made in the study of 
isolated strips of heart muscle. The less com- 
plicated geometry and the element of control 
(shortening vs. tension) possible has afforded 
the opportunity to examine more precisely the 
kinetics of contracting myocardium with a view 
to a better understanding of changes in con- 
tractility. 

Simultaneous length-tension measurements 
were made in dog and cat papillary muscle. It 
was found that in myocardium the intensity of 
active state can increase, the force-velocity curve 
can be changed, and thus the intrinsic rate of 
contraction changed in accord with varying con- 
ditions, allowing increased force of contraction 
without systolic prolongation. Force velocity 
and maximal work curves shifted to the right 
with increased initial length, increased Ca++ and 
norepinephrine ; to the left with increased K+ or 
acetylcholine. Using a modified quick release 
method (Eitchie), three phases of active state 
were determined: 1) Intensity, by redeveloped 
dp/dt. 2) Duration of Full Intensity, by de- 
parture of redeveloped dp/dt from initial dp/dt. 
3) Decay, by decline of redeveloped dp/dt. In- 
creased initial length, increased Ca++ and de- 
creased K+ increased intensity without essential 
change in duration of active state. Increased 
rate and norepinephrine increased intensity while 
shortening duration. Decreased temperature 
(30°-15° C.) prolonged duration and decay with 
little change in intensity. Dichloroisoproterenol 
blocked norepinephrine effects, but not rate stair- 
case or Ca++ effects. These data demonstrate 
that in myocardium, unlike skeletal muscle, vari- 
ations in the intensity and duration of active 
state are experimentally dissociable. 

Shortening and Tension Development in Heart 
Muscle 

It is hoped that the less complicated geometry 
of the isolated heart muscle strip will make it 
possible to gather data on the relation of myo- 
cardial oxygen consumption to tension in a situa- 
tion in which the undetermined variable due to 



NATIONAL HEART INSTITUTE 



93 



radius of curvature (La Place) in the intact ven- 
tricle would not be present. Progress has been 
blocked by the lack of a sufficiently stable and 
sensitive p0 2 electrode. It now appears that 
this instrumentation problem has been overcome. 



LABORATORY OF KIDNEY AND 
ELECTROLYTE METABOLISM 

The mechanism of bicarbonate reabsorption in 
Necturus is being reinvestigated. It is now gen- 
erally assumed that bicarbonate reabsorption in 
both the proximal and distal segments of the 
kidney is effected by an exchange of hydrogen 
ions derived from the tubular cells for intra- 
luminal sodium ions with the resultant formation 
of carbonic acid in the tubule lumen. The car- 
bonic acid is thought to be converted to C0 2 and 
water, the C0 2 escaping by diffusion. It is im- 
plicit in the theory that the rate of formation of 
C0 2 is equal to the rate of bicarbonate reabsorp- 
tion. It has been pointed out that the intra- 
luminal concentration of H2CO3 necessary to 
provide the driving force for the noncatalyzed 
dehydration of H 2 C0 3 to C0 2 and H 2 at the 
required rate would result in an intraluminal pH 
approximately 1 unit less than that when equi- 
librium is achieved. Alternatively were the re- 
action catalyzed by carbonic anhydrase residing 
in the luminal membrane the in situ pH would 
not necessarily be depressed but administration 
of carbonic anhydrase inhibitors should lower 
rather than elevate it. 

It is not possible to evaluate these alternative 
possibilities by determining the pH of fluid re- 
moved from the tubule lumen for measurement, 
since subsequent equilibration of C0 2 and H 2 - 
C0 3 in the measuring receptacle by noncatalytic 
means would raise the final pH and mask any 
in situ differences. For these reasons much of 
the effort in the past year has been directed to- 
wards the development of an appropriate micro 
pH electrode which would permit the in situ 
determination of pH in the proximal tubule fluid 
of Necturus. Thus far this has proved to be 
difficult and only a few pH-sensitive micro elec- 
trodes have been developed. In preliminary 
studies, no clearcut evidence of acidification in 
the proximal tubule of Necturus has been ob- 
served, suggesting that the dynamic pH (in situ) 



does not appreciably differ from the equilibrium 
pH. If confirmed, this observation would lend 
support to the view that the dehydration of car- 
bonic acid within the tubule lumen is catalyzed 
by carbonic anhydrase residing in the tubule cell 
membrane. 

Certain aspects of phosphate transport in the 
dog kidney, a process intimately related to uri- 
nary acidification, are being reinvestigated. Al- 
though net phosphate secretion in the chicken has 
been previously reported from this laboratory, it 
had generally been agreed that phosphate is not 
secreted in either dog or man. In recent years, 
however, several studies have appeared in the 
literature in which net secretion of phosphate 
was reported to have occurred in the dog. Since 
in none of these studies was the filtered phos- 
phate load maintained at a reasonably constant 
level, the evidence is inconclusive. In order to 
establish the presence or absence of net phosphate 
secretion in the mammal, the renal excretion of 
phosphate is being studied under steady state 
conditions in a variety of experimental situa- 
tions which include most of those previously re- 
ported to have been associated with the observa- 
tion of net phosphate secretion. Neither prior 
administration of parathormone for a variable 
period of time, prior administration of para- 
thormone and superimposed manhitol diuresis, 
prior loading with sodium phosphate for three 
days before study, or acute lowering of the glom- 
erular filtration rate, has resulted in the excre- 
tion of phosphate in excess of that filtered. 

An investigation of the effects of the infusion 
of ammonium salts on electrolyte excretion in the 
kidney has been completed. It had been ob- 
served that the administration of ammonium 
chloride into the renal portal venous circulation 
of the chicken resulted in a predominately uni- 
lateral increase in sodium chloride and water ex- 
cretion on the injected side, as well as a rise in 
the excretion of ammonia. This effect has been 
shown to be due to the ammonium ion itself, 
since it occurs with the ammonium salts of sul- 
fate, acetate, nitrate, as well as chloride. Equi- 
valent acidosis produced by hydrochloric acid 
does not result in similar changes in urinary 
composition. The natriuresis is also independent 
of the rate of ammonia excretion. It occurs when 
the ammonia excretion is minimized by prior 
alkalinization of the urine. Although in all 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



studies net potassium secretion diminished in 
association with the fall in sodium reabsorption, 
no change in net hydrogen ion transport was ob- 
served. The mechanism of the response to am- 
monium salts is unclear. It is possible that the 
effect is secondary to interference with potassium 
uptake by the renal tubule cells. NH 4 + ion may 
substitute for K+ on the contraluminal Na-K 
exchange pump. It should be noted that sub- 
stitution of ammonium ion for potassium on the 
sodium-potassium exchanger has been suggested 
on the basis of analogous studies in the red cell 
in other laboratories. 

A number of studies of the mechanism of 
urinary dilution and concentration are in prog- 
ress. The effect of one of the benzothiadiazine 
diuretics, chlorothiazide, on urinary dilution and 
concentration in the dog has been investigated. 
These agents diminish the rate of free water ex- 
cretion during water diuresis in both man and 
dog. It has been assumed that this is a conse- 
quence of interference with the reabsorption of 
sodium and chloride in a water-impermeable seg- 
ment of the renal tubule. No analogous studies 
during the elaboration of hypertonic urine have 
been reported. The present studies have also in- 
dicated that the decrement in solute-free water 
excretion when chlorothiazide is administered 
can be quantitatively accounted for by the incre- 
ment in NaCl excretion suggesting that the major 
site of action of this drug is at the site within 
the renal tubule at which removal of sodium 
and chloride leads to dilution of the urine. 

The relationship between negative free water 
and solute excretion over a wide range of solute 
excretion has been examined in hydropenic ani- 
mals pretreated with vasopressin. Results ob- 
tained during the administration of chlorothia- 
zide have been compared with those during the 
injection of nonabsorbable solute such as manni- 
tol. At low rates of solute excretion, negative 
free water clearance is unaltered by chlorothia- 
zide. In contrast, at high rates of solute excre- 
tion, a uniform increase in negative free water 
clearance occurs when chlorothiazide is admin- 
istered. Since massive solute diuresis induced by 
nonpharmacologic agents is associated with a pro- 
gressive fall in negative free water clearance, the 
rise observed with chlorothiazide under these cir- 
cumstances may be indicative of the delivery of a 
more concentrated solution from the distal con- 



volution to the terminal concentrating site. In 
this view the drug does not exert a primary effect 
on the terminal concentrating mechanism. 

The effects of this agent in patients with neph- 
rogenic diabetes insipidus have also been exam- 
ined. It had previously been reported that the 
benzothiadiazine diuretics not only increased 
urine osmolality in patients with diabetes in- 
sipidus but also diminished urine flow. Although 
the increase in urine osmolality is clearly the 
result of a diminution in sodium transport in the 
distal convolution (see above) this single action 
can not account for the associated antidiuretic 
response. Oral administration of either chloro- 
thiazide or hydrochlorothiazide to four patients 
with vasopressin resistant diabetes insipidus re- 
sulted in a uniform increase in urine osmolality 
though never to a concentration exceeding that of 
plasma, and a 3CM:0 percent diminution in urine 
flow. The effect was considerably greater when 
the patients were maintained on a low sodium 
intake than when maintained on a high sodium 
intake. Furthermore, the antidiuretic response 
could be maintained despite withdrawal of the 
drug if the restricted sodium intake was con- 
tinued throughout the course of the study. A re- 
versal to the polyuric phase was uniformly ob- 
served when sodium was readministered in the 
diet. On the basis of these observations, it has 
been suggested that antidiuresis produced by 
chlorothiazide is a consequence of a reduction in 
body sodium content. The fall in sodium con- 
tent reduces volume flow to the distal portion of 
the nephron either by a reduction in filtration 
rate or by an increase in proximal reabsorption. 
If this interpretation is correct, salt depletion 
produced by other agents should also result in 
an antidiuretic response in these subjects. This 
possibility is being examined at present. 

In association with the above studies, the effect 
of chlorothiazide has been examined in adrenalec- 
tomized rats. It has previously been suggested 
by others that the antidiuretic and chloruretic 
effect in diabetes insipidus is secondary to inter- 
ference with the renal action of aldosterone. The 
conclusion is based largely on the report that 
chlorothiazide is ineffective in adrenalectomized 
rats. In contrast, the present studies indicate 
that the diuretic response does not differ in nor- 
mal animals and in adrenalectomized animals 
maintained on an adequate salt intake. 






NATIONAL HEART INSTITUTE 



95 



Further experiments relating to the concen- 
trating mechanism are being pursued in an at- 
tempt to develop a method for the estimation of 
renal medullary blood flow in the intact animal. 
It has been established that medullary and papil- 
lary tissue of the kidney is hypertonic to plasma. 
The maintenance of this concentration gradient 
within the medullary and papillary tissue is de- 
pendent upon the unique anatomical arrangement 
of the vascular tree within the deeper portions 
of the kidney, sequestered solute being effected by 
countercurrent exchange of water and solute be- 
tween the opposing limbs of the vessel. In order 
to measure one of the variables affecting the effi- 
ciency of the countercurrent exchanger and thus 
the concentration of the urine, the possibility of 
applying a modified Fick principle for the esti- 
mation of medullary flow is being investigated 
utilizing hydrogen as an indicator. 

Net water movement and the unidirectional 
fluxes of tritiated water through a mesityl oxide 
membrane have been measured. In contrast to 
prediction, the flux ratio of water through mesityl 
oxide, a presumably nonporous organic mem- 
brane, deviates significantly from the activity 
ratios. The possibility of droplet movement 
through the mesityl oxide has been eliminated by 
comparing the flux of sodium 24 with that of 
water. Since it is apparent that neither active 
transport of water nor bulk flow occurs through 
this membrane, the discrepancy between the flux 
ratio and activity ratio is inexplicable though it 
points up the difficulty of accepting the usual 
criteria for bulk movement at face value. Pre- 
liminary measurements of the water content of 
the mesityl oxide equilibrated with water of vari- 
ous activities have indicated a significant de- 
parture from linearity between the water activity 
and the water content of the equilibrated mesityl 
oxide. If this nonlinearity is assumed to persist 
at the two interfaces of the model membrane this 
may explain the unexpectedly high flux ratio ob- 
served. 

Studies of water and solute movement through 
toad bladder have been initiated using isolated 
halves of toad bladder suspended as closed sacs 
in test solutions. In the absence of vasopressin 
net water movement in response to an osmotic 
gradient established with sodium chloride, man- 



nitol or urea, is approximately equal. In the 
presence of vasopressin, however, net transfer of 
water from a dilute urea solution within the 
bladder is greater than from comparable solu- 
tions of mannitol or sodium chloride. Further- 
more, net water gain into hypertonic solutions of 
urea is less than into equivalent concentrations 
of mannitol or sodium chloride. These differ- 
ences are dependent upon the associated rapid 
penetration of urea through the bladder wall 
presumably via aqueous pores in contrast with 
the minimal penetration of either mannitol or 
sodium chloride. 

Studies concerning the mechanism of active ca- 
tion transport in human red blood cell ghosts 
have been continued, correlating the activity of 
the sodium pump with the activity of a specific 
membrane, "ATPase." The latter is presumed 
to be involved in some fashion in the active trans- 
port of cations. The effect of various substrates 
and other agents on the ATPase activity (as 
measured with isolated membranes permeable to 
ATP) has been compared with active sodium 
transport (measured in reconstituted ghosts) . It 
has been observed that both sodium extrusion, an 
estimate of pump activity, and ATPase activity, 
specifically require ATP. Other high energy 
phosphate compounds were ineffective in both 
systems. Both sodium transport and ATPase 
activity were inhibited by sodium fluoride and 
copper chloride, but neither was affected by iodo- 
acetate, sodium azide or sodium arsenate. The 
action of strophanthidin was similar in both sys- 
tems. Both systems require magnesium and cal- 
cium inhibits both the ATPase activity and 
sodium extrusion. These comparative studies 
provide further evidence in support of the view 
that an ATPaselike enzyme residing within the 
cellular membrane is involved in the active trans- 
port of sodium and potassium across the red cell 
wall. 

The characteristics of an ATPase isolated from 
the renal cortical tissue of dog, guinea pig and rat 
are being studied. Thus far the studies are in 
a preliminary form and the precise conditions for 
maximal activation of the enzyme system have not 
yet been developed. 

A reliable automatic coulometric-amperometric 
titration method for chloride analysis has been 
developed ; in the past year the method has been 



96 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



further improved. The analysis of chloride in 
a large variety of biological fluids has been under- 
taken in order to demonstrate its validity as a 
useful biochemical method. It has been applied 
in a project designed to characterize the distribu- 
tion of chloride across tissue cells in nephrec- 
tomized rats. According to the simplified Conway 
hypothesis, appreciable amounts of chloride 
should shift into the muscle cell in the presence 
of hyperkalemia and acidosis. To test this hy- 
pothesis the distribution of chloride and of iso- 
topically labeled inulin has been compared in 
nephrectomized rats with severe acidosis and 
hyperkalemia. In the two studies completed the 
ratio of inulin to chloride spaces was 0.95 in the 
hyperkalemic animal, a value not significantly 
different from that observed in muscle tissue of 
normal rats. According to the Conway hypothe- 
sis the ratio should have fallen to about 0.75 in 
the presence of the observed degree of hyper- 
kalemia. 

An analysis of the myocardial chloride content 
in experimental cardiac failure in the dog has 
been completed. The increment in water and 
chloride content in heart muscle of dogs with 
congestive heart failure can be accounted for 
solely on the basis of an expansion of the extra- 
cellular space of this tissue. It is apparent that 
these alterations in water and electrolyte content 
do not constitute the primary defect responsible 
for the heart failure though they may in all 
probability contribute to a further depression in 
myocardial function. 

An infinite thickness liquid counting method 
utilizing the gas flow detection chamber has been 
developed in order to facilitate the estimation 
of C 14 labeled inulin. Data obtained using this 
method have confirmed results obtained in this 
laboratory a number of years ago which provided 
evidence that inulin clearance does not vary with 
changes in plasma inulin concentration; the use 
of radioactive inulin has extended the range of 
plasma concentration an additional order of mag- 
nitude lower. 

Investigation of the role of a cardioglobulin 
system in mammalian plasma has been continued. 
It had previously been reported that cardioglob- 
ulin exerts a positive inotropic effect on the iso- 



been shown that the cardioglobulin content of 
human plasma is elevated in hypertensive disease, 
markedly depressed in some patients with idio- 
pathic myocarditis, and may be acutely lowered 
during the course of extracorporeal perfusion of 
blood. The present efforts are directed at deter- 
mining the role of this protein system in the 
mammal. For this purpose the cardiovascular 
effect attendant upon depletion of cardioglobulin 
in the rat has been assessed. Chronic cardioglob- 
ulin depletion by exchange transfusion has proved 
virtually impossible, as a result of the extreme 
rapidity of the regeneration of the protein. Al- 
though it has been possible to demonstrate an 
acute diminution in cardioglobulin concentration 
in rat plasma, a return to normal values occurs 
with 24 hours. The acute cardiovascular effects 
of administration of cardioglobulin-free plasma 
to the rat have thus far proved to be negligible, 
in part due to methodological difficulties. In 
acute studies administration of cardioglobulin 
rich plasma to the failing rat heart-lung prepa- 
ration does not affect cardiac performance as 
estimated by alterations in heart rate, blood pres- 
sure, blood flow through a shunt and venous 
pressure. Further experiments are planned in 
order to verify this conclusion. 

The mechanisms regulating the secretion and 
metabolism of aldosterone in dogs with secondary 
hyperaldosteronism have been studied. A reex- 
amination of the role of the arterial baroreceptor 
in the control of aldosterone secretion has been 
completed. On the basis of these studies, it may 
be concluded that the baroreceptors in the aortic 
arch and the carotid arterial tree are not essential 
to either the hypersecretion of aldosterone or the 
almost complete sodium retention observed in dogs 
with inferior vena caval constriction, and fur- 
ther, that marked reductions in pulse pressure in 
the carotid arterial system and in the mesenteric 
arterial tree fail to increase the rate of aldoster- 
one secretion. More recently, in collaboration 
with Drs. Anderson, Haymaker and Spense of 
NIAMD, a role of the midbrain in the regulation 
of aldosterone excretion in the same experimental 
preparation has been excluded. Thus, following 
midbrain transaction, acute hemorrhage results 
in a striking increase in adrenal vein aldosterone 



lated frog heart. Using this as an assay it has secretion in dogs with vena caval constriction. 



NATIONAL HEART INSTITUTE 



97 



The response does not differ appreciably from 
that observed in vena caval dogs with intact 
midbrains. It has also been shown that the 
adrenal and kidney transplanted to the cervical 
region, though devoid of nervous connections, re- 
spond in the "normal" fashion to acute hemor- 
rhage. Aldosterone secretion increases and sodium 
retention supervenes in the cervically transplanted 
organ, despite an absence of both nervous con- 
nections and an increase in venous pressure in 
these organs. 

The most interesting observation made in the 
past year in relation to this problem has been the 
demonstration that the kidney releases an aldos- 
terone stimulating hormone which signals the 
release of mineralocorticoids from the adrenal. 
In the absence of ACTH, enhanced secretion of 
aldosterone from the adrenal in dogs following 
acute hemorrhage is considered presumptive evi- 
dence for secretion of aldosterone secreting hor- 
mones. In a series of successive ablation experi- 
ments, removal of the anterior pituitary, removal 
of the head, of the liver, etc. was generally fol- 
lowed by an increase in aldosterone secretion in 
response to acute hemorrhage. In contrast, re- 
moval of the kidneys prevented the increase in 
aldosterone secretion generally attendant upon 
acute blood loss. Furthermore the intravenous 
infusion of saline extracts of kidney resulted in 
a marked increase in aldosterone production. 
These data provide conclusive evidence for the 
renal origin of an aldosterone stimulating hor- 
mone. 

The aldosterone stimulating hormone derived 
from kidney does not appear to be renin. Experi- 
mental hypertension in the dog is not associated 
with increased rates of aldosterone secretion. 
Kenin administration does not augment aldos- 
terone secretion although injection of hypertensin 
II may in some instances stimulate its release. 
An associated increase in corticosterone secretion 
due to hypertensin II, not generally seen follow- 
ing hemorrhage in vena caval dogs, suggests that 
the response to hypertensin II is not analogous 
to that due to aldosterone stimulating hormone 
of renal origin. More recently it has been shown 
that there is a marked fall in aldosterone secre- 
tion following nephrectomy in hypophysectom- 
ized dogs with constrictions of the inferior vena 
cava. This is additional support for the role of 
the kidney in the elaboration of a tropic hormone. 



LABORATORY OF CLINICAL 
BIOCHEMISTRY 



Amine Biogenesis and Metabolism 

It is now certain that there is one enzyme, 
designated as aromatic L-amino acid decarboxyl- 
ase, that is responsible for decarboxylation of 
all the normally occurring aromatic L-amino 
acids including 3,4-dihydroxyphenylalanine, 5- 
hydroxytryptophan, tryptophan, tyrosine, phe- 
nylalanine, histidine and kynurenine. Substrate- 
enzyme studies have been carried out for all these 
amino acids. In addition to this general amino 
acid decarboxylase there is also found a specific 
L-histidine decarboxylase. This means that there 
are at least two different catalysts for converting 
the dietary amino acid histidine to the potent 
pharmacologic agent histamine. In comparing 
the two mechanisms for histidine decarboxyla- 
tion, it was found that the specific enzyme is 
present in tissues that are rich in mast cells (mast 
cell tumors) ; it acts only on L-histidine, has the 
higher affinity for histidine (K m lO^M) and has a 
pH optimum near 6. It is not inhibited by 
a-methyl DOPA. By contrast histidine decar- 
boxylation by L-aromatic amino acid decarboxy- 
lase occurs in organs containing few mast cells, 
the K m is nearer lO^M, pH optimum is about 8.5 
and the activity is inhibited by low concentra- 
tions of a-methyl DOPA. 

The significance of these two separate mech- 
anisms for histidine decarboxylation is not ap- 
parent. However, it has been suggested by Dr. 
Richard Schayer, that there is a marked increase 
in histidine decarboxylase in some tissues (up to 
10 fold) during conditions of stress. Studies on 
the mechanism of this apparent enzyme induction 
are now in progress. 

Inhibitors of aromatic L-amino acid decarboxy- 
lase were studied further and it has been found 
that the two most potent ones, a-methyl DOPA 
and a-methyl metatyrosine, have two actions in 
animal tissues in vivo. The first action appar- 
ently results from its known enzyme-inhibiting 
activity and leads to a fall in the serotonin and 
dopamine concentration of brain. However, 24 
hours after a single dose of about 100 mg/kg the 
concentration of these two amines has returned 
to normal. On the other hand, the concentration 
of noradrenaline in heart and brain falls to values 



98 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



less than 10 percent of normal and remains low 
for periods of several days. Various experimen- 
tal procedures indicate that the a-methyl amino 
acids prevent the binding of noradrenaline by 
tissues. It would appear, therefore, that they 
act in a manner similar to reserpine with the 
important difference that their effects are limited 
to noradrenaline. This property makes them ex- 
tremely valuable as tools in studying the func- 
tions of brain amines since it is possible to prepare 
animals which have normal amounts of brain 
serotonin and dopamine but little noradrenaline. 
This is essentially the situation 24-30 hours after 
a single dose of a-methyl metatyrosine in mice, 
rats, guinea pigs and rabbits (100-200 mgAg)- 
It is of interest that after an initial short period 
of apparent sedation the animals appear perfectly 
normal. Studies on the mechanism of the anti- 
hypertensive action of these agents in man by the 
Section on Experimental Therapeutics are com- 
plementary to those in this laboratory. In addi- 
tion, members of the Laboratory of Chemical 
Pharmacology have used the a-methyl amino 
acids in their studies on the physiology of the 
amines. 

One of the consequences of the existence of a 
nonspecific L-amino acid decarboxylase is that 
amine derivatives of all of the normally occur- 
ring amino acids should be formed. This appears 
to be the case and phenylethylamine, tyramine 
and tryptamine have been shown to be excreted 
in the urine. Ortho and meta tyramimne are also 
found although it is not yet known how they are 
formed. Other interesting amines which have 
been found in urine are synephrine and nor- 
synephrine (p-hydroxyphenylethanolamine). The 
former, which may be considered as adrenaline 
with one less ring hydroxyl group, is found in 
urine in amounts which are comparable, and fre- 
quently greater, than that of the normetaneph- 
rines. Since synephrine is a fairly active phar- 
macologic agent, its presence in such amounts 
may have physiologic significance. However, of 
more biochemical interest was the indication that 
the side chain oxidation, such as occurs in the 
conversion of dopamine to noradrenaline, can oc- 
cur with other compounds. It was subsequently 
found that purified preparations of the enzyme, 
dopamine-/?-oxidase, oxidize tyramine to nor- 
synephrine and that the enzyme is therefore not 
specific for dopamine. The finding of the N- 



methylated compound in urine indicates that the 
enzyme noradrenaline N-methylpherase is also 
nonspecific. It appears, therefore, that most of 
the enzymes involved in aromatic amine biogene- 
sis are nonspecific. This nonspecificity explains 
the presence of the numerous aromatic amines 
and their metabolites in urine. 

An important route of metabolism of serotonin 
leads to the pineal gland hormone, melatonin 
(N-acetyl-5-methoxytryptamine). In conjunction 
with members of NIMH it has been shown that 
in the pineal gland serotonin is first acetylated 
and then methylated to yield melatonin. The 
two enzymes have been partially purified and 
studied. The methylpherase is specific and is 
found only in the pineal gland. Melatonin which 
leaves the pineal gland is metabolized in the liver 
by the microsomal hydroxylating system to yield 
6-hydroxymelatonin. 

Amine metabolism is also carried out by non- 
specific enzymes and many products are to be 
expected in urine. Methods for the assay of the 
o-methylated metabolites of the epinephrines and 
of their acid end product, 3-methoxy-4-hydroxy- 
mandelic acid, have been developed. These are 
specific and precise. Assays of a similar type 
are applicable to norsynephrine and p-hydroxy- 
mandelic acid. All these assays are now being 
used in collaboration with the Section on Experi- 
mental Therapeutics to study these amine path- 
ways in man, normally and in various disease 
states. In the case of noradrenaline two major 
routes of metabolism are possible, oxidative de- 
amination and o-methylation. Studies on animals 
in vivo indicate that only in the liver is o-methy- 
lation the major route of noradrenaline metabo- 
lism. In brain, heart, and other organs the major 
route is oxidative deamination. This means that 
noradrenaline synthesized and released within an 
organ is metabolized by monoamine oxidase, only 
that fraction which leaks out into the blood is 
methylated on passage through the liver. 

The enzyme, monoamine oxidase (MAO) has 
now been obtained in soluble form from mito- 
chondria and has been purified at least 10 fold 
from this source. This represents a considerable 
advance in purification. It has been found that 
the purified enzyme splits N-dimethyl amines 
into the corresponding aldehyde and dimethyl- 
amine and that N-dimethylamine oxides are de- 
aminated in the absence of oxygen. It may be 



NATIONAL HEART INSTITUTE 



99 



that MAO catalyzes a direct oxidative attack on 
the nitrogen and that the N-oxides are interme- 
diates. While a few N-oxides have been isolated 
from natural sources no enzymatic studies of this 
type have ever been carried out. Other oxidative 
deaminases such as L-amino acid oxidase, D-am- 
ino-acid oxidase and diamine oxidase are really 
dehydrogenases, the oxygen coming from hydroly- 
sis of an intermediate imino compound. In the 
case of MAO, oxidation must then occur through 
atmospheric oxygen. Experiments with O 18 are 
now in progress to verify this point. 

Choline Biogenesis 

The biogenesis of choline has been studied in 
animals. Although serine is the ultimate pre- 
cursor it was not possible to demonstrate direct 
decarboxylation of this amino acid to yield free 
ethanolamine. These studies indicated, rather, 
that serine is incorporated into phospholipid and 
in this form is decarboxylated to phospholipid- 
ethanolamine (cephalin) and, still in lipid form, 
is methylated through intermediate mono and 
dimethyl forms to phospholipid choline (leci- 
thin). All the methyl groups arise from the 
methyl groups of methionine. Utilization of one 
carbon sources such as formate or formaldehyde 
occurs only by prior conversion to methionine 
methyl groups. Overall synthesis of choline from 
serine, in phospholipid form, also follows from 
the work of Dr. David Greenberg and collabo- 
rators at the University of California. Such a 
pathway may have great physiologic significance. 
Conversion of a dimethylamine to a quarternary 
amine in a phospholipoprotein structure would 
be a most effective way to alter the charge on a 
membrane and thereby regulate transport mech- 
anisms. In liver the entire series of reactions 
occurs in the microsomal portion of the cell. It 
may be that they are involved in determining 
the lamellar structure of the endoplasmic re- 
ticulum. 

Aminobutyric Acid Metabolism 

Further studies on y- am i n °butyric acid 
(GABA) metabolism were carried out. The ma- 
jor work has been on the identification of the 
GABA-histidine containing peptide in brain as 
y-aminobutyryl histidine (homocarnosine). It 



represents the GABA analogue of carnosine. The 
latter is found mainly in muscle and other 
peripheral organs and does not occur in brain. 
Homocarnosine is found only in the central ner- 
vous system and urine. Methods for assay of 
the several carnosines have been developed and 
brain levels have been measured in many animal 
species. In man, monkey and cattle, brain con- 
tains as much as 30 ^g/gram. Although this 
is considerable it represents only 5 percent of 
the total GABA content. The unique localization 
would appear to be indicative of the availability 
of /3-alanine or GABA in the various tissues 
rather than of enzyme specificity. When sig- 
nificant GABA levels were maintained in peri- 
pheral organs (by including GABA in the diet) 
appreciable amounts of homocarnosine appeared 
in muscle. Normally it is not found there. These 
findings corroborate experiments of Meister con- 
cerning the lack of specificity of the enzyme, 
carnosine synthetase. The significance of homo- 
carnosine in brain remains to be determined. Its 
presence in urine may provide another measure 
of central nervous system chemistry since it is 
normally formed only in brain. 

Amino Acid Transport 

Studies on the uptake of aromatic amino acids 
by various tissues in vivo and in vitro have been 
continued. L-Tyrosine uptake by brain in vivo 
unquestionably involves a mechanism of facili- 
tated transport. It has now been found that it 
has a most rigid stereospecificity. In previous 
studies it was found that following administra- 
tion of D-tyrosine some tyrosine appeared in 
brain, though much less than with L-isomer. It 
has now been found that the tyrosine entering 
brain after administration of D-tyrosine is all in 
the L-form indicating that racemization had 
taken place before penetration into brain. The 
D-form does not pass the "blood brain barrier." 
Little or no distinction between uptake of D and 
L-tyrosine occurs in other tissues. These and 
other findings suggest that the same anatomical 
area which is considered as the "blood brain bar- 
rier" also possesses mechanisms to facilitate the 
transport of essential metabolites of the central 
nervous system. Such findings make it necessary 
to point out what should have been obvious be- 
fore this, that the "blood brain barrier" theory 



100 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



requires the existence of mechanisms of active 
transport at the same site, to explain the uptake 
of metabolites with physical properties com- 
parable to those which are usually prevented 
from penetrating. Although much of the mecha- 
nism for facilitated transport of L-tyrosine re- 
sides in the "blood brain barrier" and can be 
demonstrated only in the intact animal, brain 
slice uptake of tyrosine is also unique. Brain 
slices concentrate tyrosine as much as 3 fold over 
the incubation medium. To do this they require 
an energy source which can be supplied by glu- 
cose, mannose and related compounds. Meta- 
bolic poisons inhibit this uptake. Muscle, liver, 
spleen and kidney slices do not concentrate tyro- 
since. In rat diaphragm muscle all the uptake 
of L-tyrosine can be explained on a diffusion 
mechanism. These differences between brain and 
other tissues in uptake of L-tyrosine no doubt 
extend to many other amino acids and metabo- 
lites and it will be of interest to examine other 
metabolites in the same way. 

Mechanisms of Aromatic Hydroxylation 

Some additional studies have been carried out 
on the mechanism of aromatic hydroxylation. 
Using tritium labelled phenylalanine (ring) and 
T 2 it was found that the hydrogen in the para 
position of phenylalanine does not become la- 
bile in the presence of phenylalanine hydroxy- 
lase. An intermediate substrate activation 
should have yielded exchange between the para 
hydrogen of phenylalanine and water. This did 
not occur. Similar studies are being started with 
acetanilide labelled with tritium in the para posi- 
tion. In this case microsomal aromatic hydroxy- 
lase will be used. 

Collagen and Hydroxyproline 

Studies on collagen biosynthesis and hydroxy- 
proline formation have been extended. The pres- 
ence of rapidly turning over forms of collagen, 
suggested by studies on urinary hydroxyproline 
have been confirmed by direct studies on tissue 
collagen. This confirms our previous conclusion 
that although body collagen as a whole is meta- 
bolically inert there are small pools of collagen 
which are in the dynamic state as are other tis- 
sue constituents. Some of these findings are being 



explored in man by the Section on Experimental 
Therapeutics. Studies on chick embryos have 
shown that collagen (hydroxyproline peptide for- 
mation) occurs in microsomes and its require- 
ments are similar to those of other protein syn- 
theses. In preliminary studies it has been possible 
to demonstrate "collagen" formation in vitro in 
microsomal material isolated from chick embryos. 
Studies have been initiated to investigate the 
metabolic relationship between proline, hydroxy- 
proline and ketoproline found in the various 
actinomycin molecules. Micro-procedures for the 
isolation of the different actinomycins were de- 
vised. In an initial study with C 14 labeled L-pro- 
line it was found that the actinomycins synthe- 
sized by washed suspension of 8. antibioticus 
were isotopically labeled. Moreover, hydroxy- 
proline was found to contain appreciable radio- 
activity. Formation of the actinomycin chromo- 
phore, 2-amino-4,6-dimethyl-3-phenoxazinone-l,9- 
dicarboxylic acid, has been achieved using 
cell-free extracts of S. antibioticus and 3-hy- 
droxy-4-methylanthranilic acid as substrate. In 
addition, the cell-free system will condense a 
number of other ortho-aminophenols for example, 
3-hydroxykynurenine, 3-hydroxy-4-methylanthra- 
nilic acid methylester and 3-hydroxyanthranilic 
acid to form phenoxazones. The purification and 
properties of this enzyme system are now under 
investigation. It may be possible to achieve an 
overall enzymatic synthesis of the proline and 
hydroxyproline actinomycin chromopeptides. 



GENERAL MEDICINE AND EXPERIMENTAL 
THERAPEUTICS BRANCH 



Section on Clinical Endocrinology 

The research program of the Section on Clini- 
cal Endocrinology has included studies on (1) 
aldosterone metabolism in edematous states, 
(2) calcium metabolism in metabolic bone dis- 
ease, (3) renal function, with special reference to 
free water clearance, and (4) experimental 
atherosclerosis. 

(1) Studies on aldosterone metabolism included 
clinical studies on the role of aldosterone in fast- 
ing, in primary and secondary aldosteronism, in 
idiopathic edema, and in potassium depletion, 



NATIONAL HEART INSTITUTE 



101 



both spontaneous and experimentally induced. 
The role of the kidney and of various pressor 
agents in the regulation of aldosterone secretion 
has been explored in dogs. 

It was confirmed that the sodium loss with 
fasting is greater than that with sodium depriva- 
tion alone; in some patients this was associated 
with failure of aldosterone secretion to rise with 
salt loss, but these patients lost potassium with 
fasting, and the relative hypoaldosteronism may 
have been a result of this. Eight patients with 
primary aldosteronism were studied before and 
after surgery. The use of a low sodium diet pre- 
operative^ allowed restoration of potassium defi- 
cits and an increase in aldosterone secretion. The 
same result was seen with aldosterone antagonists, 
which thus might increase aldosterone secretion 
in primary aldosteronism (by promoting potas- 
sium retention) as well as in secondary aldos- 
teronism (by inducing sodium loss). 

The expansion of intravascular volume with 
albumin, on the other hand, did not lower aldos- 
terone secretion in primary aldosteronism as it 
does in secondary aldosteronism. Direct meas- 
urements of blood volume in patients with edema 
showed an inverse relationship of intravascular 
volume and aldosterone secretion; a similar in- 
verse relationship related arterial pulse pressure 
and aldosterone secretion. Methodology for de- 
termination of aldosterone was improved by in- 
troduction of C14 aldosterone biosynthetically 
prepared, and of a "visible" aldosterone marker. 
Studies on gas chromatography of aldosterone 
were initiated. 

A study of the possible role of diurnal rhythms 
in the cause of edema was instituted by extensive 
"mapping" of diurnal rhythms in normal female 
subjects. Aldosteronism was found associated 
with hyperplasia of juxtaglomerular apparatus 
in a normotensive boy, and the role of the kidney 
in control of aldosterone secretion was studied 
in extenso in dogs. Nephrectomy was found to 
reduce or abolish the response of aldosterone 
secretion to caval constriction in the hypophysec- 
tomized dog. Contrary to reported findings, this 
response was found to occur in hypophysecto- 
mized dogs both with and without suprapontine 
brain removal. Hypertensin (Angiotensin II) 
and renin were shown to induce aldosterone se- 
cretion in the hypophysectomized, nephrecto- 
mized animal, and did so in some preparations. 



(2) Studies on calcium metabolism and meta- 
bolic bone disease included clinical balance 
studies in osteoporosis, in renal osteitis, in sar- 
coidosis, and in hyperparathyroidism, studies on 
renal phosphate clearance with special reference 
to parathyroid function, studies of labile calcium 
in the parathyroidectomized dog, and studies in 
the metabolism of vitamin D. 

Patients with idiopathic osteoporosis (defined 
as "idiopathic" by an absence of the response of 
calcium balance to estrogens or androgens) were 
studied to ascertain the mode of action of albu- 
min in inducing positive calcium balance and to 
test the effect of strontium. The possibility of a 
purely oncotic action of albumin could not be 
ruled out; strontium appeared to be without 
effect. Patients with renal osteitis were found to 
absorb little or no calcium from the gastrointes- 
tinal tract, as judged from balance studies and 
the fecal excretion of orally fed calcium 47 . Vita- 
min D markedly improved calcium absorption, 
but aluminum hydroxide did not. 

Patients with sarcoidosis were found to absorb 
abnormal amounts of calcium from the gastro- 
intestinal tract, to increase this absorption fur- 
ther with vitamin D, and to lower it markedly 
with carbohydrate-active corticosteroids even in 
the presence of vitamin D. Blood levels of vita- 
min D were never elevated, suggesting hypersen- 
sitivity to, rather than hyperabsorption of, vita- 
min D. 

Renal phosphate and calcium excretion were 
studied in patients with hyperparathyroidism 
and in normal control subjects. Normal subjects 
could not be distinguished from those with hyper- 
parathyroidism on the basis of either maximal 
reabsorption or clearance of phosphorus, but 
phosphate excretion in response to a calcium load 
was abnormal in hyperparathyroidism, as was 
renal calcium excretion on very low phosphorus 
intake. Both phenomena could be reproduced 
in normal subjects with chronic administration 
of parathyroid extract. 

The role of the parathyroids in the renal ex- 
cretion of phosphate was studied with clearance 
techniques in dog and man. No evidence for 
tubular secretion of phosphate could be found, 
despite phosphorus loading, acidosis, and use of 
parathyroid extract. Clearcut tubular maxima 
were found, and considered to be evidence against 
the possibility of secretion. 



102 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Parathyroidectomized dogs were found to have 
decreased amounts of readily labile bone calcium, 
as judged from dynamic response of extracellular 
fluid calcium to versene-induced hypocalcemia. 
Parathyroid extract and vitamin D could restore 
labile calcium to normal and, with higher doses, 
to above normal. H 3 vitamin D was prepared 
for us by a commercial firm, and C14 vitamin D 
will be prepared in the near future. Chromato- 
graphic systems for isolation of vitamin D have 
been developed, and in vivo and in vitro studies 
of its metabolism and fate have been begun. 

(3) Studies on renal function included meas- 
ures of free water clearance in patients with 
edema and in patients with hyponatremia, and 
studies with Amphotericin B. 

Free water excretion was studied in patients 
with cirrhosis and compared with that in pa- 
tients with cardiac failure and in normal subjects 
depleted of sodium. Results indicate that in all 
these conditions free water excretion is limited, 
not because there is persistent or excessive secre- 
tion of antidiuretic hormone, but because there is 
excessive reabsorption of salt and water in the 
proximal tubules. "Whereas this appears to be 
aldosterone-dependent (as judged from the re- 
sponse to aldosterone antagonists) in the patients 
with cirrhosis as in the normal subjects, it ap- 
pears not to be aldosterone-dependent in some 
patients with cardiac failure: in these patients, 
the filtration fraction appeared to be above nor- 
mal: catechol amine antagonists had no effect in 
decreasing proximal sodium reabsorption. 

Further studies were carried out in the syn- 
drome of hyponatremia resulting from "inappro- 
priate" secretion of antidiuretic hormone. The 
syndrome was uncovered in two patients with 
intermittent acute porphyria. Some fifteen cases 
with pulmonary or intracranial diseases have 
been uncovered in various clinics and are the 
subject of a review in preparation. The syn- 
drome could be reproduced with pitressin in nor- 
mal subjects, either by deliberate choice of fluid 
intake or by voluntary ad libitum drinking. 

Amphotericin B was found to induce reversible 
decrease of glomerular filtration rate and renal 
plasma flow in patients receiving the drug for 
therapeutic purposes. 

(4) Studies in experimental atherosclerosis in- 
cluded measures of the rate of penetration of 
labeled cholesterol and of labeled albumin into 



the aorta of the dog. It was found that both 
substances entered more rapidly in the proximal 
and progressively less rapidly down the distal 
aorta. The gradient for albumin does not depend 
upon pulsatile pressure or upon absolute circum- 
ferential tension, but the rate does appear to be 
pressure-dependent. Further studies, including 
measurement of lipoprotein in the aortic wall, 
are in progress. 

Section on Experimental Therapeutics 

The investigative approach of this laboratory 
to clinical cardiovascular problems is a combined 
biochemical-pharmacologic one. For convenience 
the studies are described under four headings: 
(1) biogenic amines, (2) amino acid metabolism, 
(3) action and metabolism of drugs, and (4) mis- 
cellaneous. 

Biogenic Amines 

Using improved methods, the urinary excretion 
of catecholamines (norepinephrine plus epineph- 
rine), their methoxy-amine metabolites (nor- 
metanephrine and metanephrine) and 3-methoxy- 
4-hydroxymandelic (MOMA) was measured in 
23 patients with pheochromocytoma and in a 
large group of patients with essential hyperten- 
sion. For purposes of separating patients with 
pheochromocytoma from the rest of the hyper- 
tensive population, a suitable upper limit of nor- 
mal for each assay was found to be 0.1 mg/day, 
1.3 mg/day and 6.0 mg/day respectively. It was 
determined that the production of catecholamines 
as indicated by urinary products is not elevated 
above normal in essential hypertension. 

The fact that the excreted methoxy-amines rep- 
sents a much smaller fraction of the metabolites 
in normals and hypertensives than does MOMA 
supports the idea that oxidative deamination is 
the initial mode of metabolic degradation of that 
norepinephrine formed at nerve endings. Studies 
in the rat tend to support this concept. Mono- 
amine oxidase activity of brain and heart was 
found to be several times that of catechol-0- 
methyltransferase. Endogenous norepinephrine 
accumulated when monoamine oxidase was inhib- 
ited by drugs; exogenously administered norepi- 
nephrine accumulated in the heart under the 
same conditions. Neither of these effects could 



NATIONAL HEART INSTITUTE 



103 



be produced by inhibition of catechol-O-methyl 
transferase. 

The vascular response to various sympatho- 
mimetic amines is being compared with the effects 
of these agents on blood unesterified fatty acid 
(UFA). While norepinephrine alters both vari- 
ables, equipressor doses of dopamine in patients 
were found not to provoke an UFA response. 

In collaboration with Dr. J. Pisano (LCB), 
N-methyl - p - hydroxy - phenylethanolamine (sy- 
nephrine) was isolated and identified in human 
urine. That tyramine may be the precursor of 
synephrine of norsynephrine is indicated by the 
finding of elevated amounts of p-hydroxy-man- 
delic acid in the urine after infusion of tyramine 
in man. The overall metabolism of tyramine and 
the physiologic significance of the synephrines 
are under investigation. 

A sensitive and specific chemical assay for his- 
tamine in human urine has been developed. Nor- 
mal values are in the range of 20 to 100 ^g/day. 
In a unique case of the carcinoid syndrome aris- 
ing from a primary lesion in the stomach, char- 
acterized chemically by the excretion of large 
amounts of 5-hydroxytryptophan, serotonin and 
5-hydroxyindoleacetic acid in the urine, urinary 
histamine was 545-580 fig/day. 

Amino Acid Metabolism 

It has been established that urinary hydroxy- 
proline (HPr) arises primarily from the break- 
down of body collagen, and that the amount 
excreted is a useful index of collagen synthesis 
and degradation. There is a progressive decrease 
in the amount of urinary HPr in successive dec- 
ades of life, presumably reflecting the "metabolic 
age" of an individual's collagen. The possible 
application of this finding to gerontologic prob- 
lems as well as to selected disorders of connective 
tissue is apparent. 

Studies of the inhibition of aromatic amino 
acid decarboxylation by a-methhyl-3,4-dihydroxy- 
DL-phenylalanine (a-methyl-dopa) have been ex- 
tended. Inhibition by this agent of the formation 
of dopamine, tryptamine, tyramine, serotonin and 
phenylethylamine from their corresponding am- 
ino acids in man has been shown. That the 
decarboxylase step of serotonin synthesis is spe- 
cifically attacked was demonstrated in two car- 
cinoid patients by the observation of an elevated 
excretion of 5-hydroxytryptophan (5HTP) co- 



incident with treatment. Administration of 
a-methyl-dopa to two patients with pheochromo- 
cytoma resulted in a slight (20 percent) but prob- 
ably significant reduction in the excretion of 
catecholamine metabolites. Decarboxylase inhi- 
bition with a-methyl-dopa was shown pharmaco- 
logically in the dog by blockade of the inotropic 
response to L-Dopa and in man by decreased 
intestinal motility response to 5-HTP. Several 
additional inhibitors have been synthesized in 
the Merck, Sharpe and Dohme laboratories and 
are in various stages of animal and clinical 
evaluation. 

Action and Metabolism of Drugs 

In studies with eight different monoamine oxi- 
dase (MAO) inhibitors, we have observed an 
orthostatic hypotensive effect rather uniformly 
in hypertensive subjects when evidence of effec- 
tive MAO inhibition was demonstrable. The lat- 
ter is indicated by increases in the urinary excre- 
tion of amines such as tryptamine and tyramine. 
Inhibitors containing the hydrazine moiety have 
frequently produced toxic effects in man at the 
doses required to lower blood pressure. Thus the 
preliminary finding that N-benzyl-N-methyl-2- 
propynyl-amine HC1 (MO-911) is an effective 
antihypertensive agent and potent MAO inhib- 
itor in man is of special interest. Also, since the 
structure of this agent is different from that of 
previous inhibitors, the evidence of a causal rela- 
tionship between MAO inhibition and blood pres- 
sure lowering seems fairly conclusive. Nonethe- 
less, the exact mechanism is undetermined. 
Neuropharmacologic experiments in dogs and cats 
have shown a sympathetic ganglion blocking ac- 
tion of several inhibitors; however, this effect 
occurs only with large doses and is rather evanes- 
cent. Possibly only a slight modification of gan- 
glionic function is sufficient to produce postural 
hypotension in man. Among other possibilities, 
peripheral and/or central accumulation of an 
amine such as dopamine could result in competi- 
tive block of norepinephrine action. 

It is claimed that MAO inhibitors benefit pa- 
tients with angina pectoris. A rational basis for 
this might be the observations in two patients 
receiving the inhibitor, isocarboxazid, of ob- 
tunded rises in pulse rate and blood pressure 
during exercise. This implies diminished cardiac 
response during exertion. 



104 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



In 40 hypertensive patients treated for six 
weeks to fifteen months with a-methyl-dopa, ini- 
tial sedation followed by subtle tranquilization 
and lowering of blood pressure which is predomi- 
nantly orthostatic have been observed rather uni- 
formly. The potency of the compound is en- 
hanced in patients with impaired renal function 
and it is uniquely effective in emergency control 
of severe hypertension particularly if used intra- 
venously. Eecent availability of the L isomer 
(Aldomet) has largely solved the problem of high 
daily intake of capsules for maintenance therapy. 
Broad evaluation of this agent as an antihyper- 
tensive is currently in progress in 16 clinics. 
Only three of eight carcinoid patients have shown 
symptomatic relief with a-methyl-dopa. Develop- 
ment of hypotension has often prevented attain- 
ment of effective dose levels. However, decar- 
boxylase inhibition appears to be a sound ap- 
proach to treatment of carcinoid, in part because 
it now seems doubtful that it is a basis for de- 
velopment of hypotensive drugs. Biochemical 
studies in rats and guinea pigs (LCB), pharma- 
cologic studies in dogs, and combined studies of 
drug metabolism and blood pressure responses in 
patients all suggest that a-methyl-dopa has an 
activity above and beyond decarboxylase inhibi- 
tion. For the moment, it is assumed that a selec- 
tive depletion of tissue stores of norepinephrine 
accounts for the lowering of blood pressure. 
Clinical studies of other decarboxylase inhibitors, 
some with and others without norepinephrine- 
depleting properties, will be initiated shortly. 

Section on Cardiodynamics 

This Section has as its ultimate objective the 
study of the biophysical and physiologic behavior 
of the cardiopulmonary system of normal and 
diseased human subjects as they go about their 
usual daily activities and are subjected to various 
physiologic, psychic, pharmacologic, and other 
stressful interventions. The measurement of a 
large number of physiologic variables under con- 
ditions most nearly simulating normal activity 
has made the development of new methods of in- 
strumentation essential. Progress in this broad 
area has and will continue to depend on a vigor- 
ous program in instrumentation development as 
well as a comprehensive study of the biomathe- 
matical models of the vascular and pulmonary 



systems. Thus, the major efforts of this Section 
have been directed along three lines: (1) instru- 
mentation, (2) biophysics of the vascular system, 
(3) biophysics of the pulmonary system. 

Instrumentation. The needs for improved in- 
strumentation have made necessary a continuous 
instrument evaluation program. Most of the 
efforts in this area have been toward the develop- 
ment of a data processing and computing unit. 
Commercially available equipment has been modi- 
fied to meet the biological and biophysical re- 
quirements of the Section. Many technical prob- 
lems have been solved so that there is now 
available an efficient, smooth working, physio- 
logical recording system which permits the ac- 
quisition of multiple channels of physiological 
information from animal or man. These data 
may be recorded on conventional direct writing 
recorders, and at the same time stored by an 
electromagnetic FM tape system. Uniquue meth- 
ods of programming the electromagnetic tape 
system to work either with an analogue to digital 
converter or with an analogue computer have 
been worked out. For example, a given time 
increment of information, such as one heart beat, 
may be searched out and programmed to play 
repetitively for study and computation. 

Pressure measurement has remained one of the 
most important unsolved problems, and therefore, 
new techniques are being sought. Several minia- 
turized pressure transducers have been investi- 
gated, one of which has shown considerable 
promise. However, none has been found suffi- 
ciently accurate for the more precise biophysical 
measurements. An extravascular pressure sensing 
device is being developed in conjunction with the 
Astra Corporation. This device can be surgically 
implanted in animals, and, it is hoped, will open 
the avenue to accurately telemetered blood pres- 
sure from living, intact, active animals. This in- 
formation has not been available previously, and 
if successful, the attempt to measure it will be 
a major step toward the accomplishment of the 
primary objective. 

The measurement of instantaneous blood flow 
in the living intact organism has remained one 
of the primary objectives. Studies in this area 
have been carried out in mechanical models and 
in the living animal. Experimental studies with 
a mechanical flow generating device have revealed 



NATIONAL HEART INSTITUTE 



105 



that the relationship between the instantaneous 
pressure gradient in a cylindrical tube and the 
instantaneous pulsatile flow is such that it may 
be predicted mathematically with reasonable ac- 
curacy. The velocity profile was examined by 
the introduction of blue dye strands into the 
pulsatile flow and indicated that in the range of 
physiologic frequencies the velocity profile across 
the tube is blunt, with most of the fluid shear 
occurring at the boundaries of flow. The more 
peripheral sleeves of flow appear to be in phase 
with the pressure gradient, while the more central 
portions of the flow appear to be in quadrature 
with the pressure gradient. This finding is quali- 
tatively in agreement with mathematical pre- 
diction. 

In the animal, blood velocity has been com- 
pared from the simultaneous recordings from a 
Kolin electromagnetic flowmeter and the com- 
puted pressure gradient technique. Agreement 
of peak blood velocities by the two methods is 
quite good. 

Cardiovascular Biophysics. The viscoelastic 
properties of the living vascular tissue have been 
studied. The results indicate that the pressure- 
diameter relationships of almost all blood vessels, 
in the systemic as well as pulmonary system ap- 
pear to be described to a first approximation as 
a simple elastic system, particularly for the lower 
frequencies of pulsation. It would appear also 
that the longitudinal motion of blood vessels may 
be neglected as a boundary condition assumption 
in the solution of the hydrodynamic equations of 
flow. 

The instantaneous blood velocity, pressure, 
pressure gradient and vessel radius are being 
measured and recorded on an electromagnetic 
tape system so that computation of the various 
terms of the Navier-Stokes equations may be ac- 
complished. Preliminary evidence indicates that 
a large majority of these terms may be neglected. 
Therefore, it is reasonable that a relatively sim- 
ple, yet realistic equation describing the pressure, 
flow, diameter relationships in the vascular sys- 
tem can be established. 

In order to study the theoretical aspects of 
heart function, it is necessary to determine the 
nature of the load against which the heart must 
work. To this end, studies are being carried out 
to examine the input impedance of the pulmonary 



artery and the aorta. This is done by measuring 
simultaneously the instantaneous blood flow and 
pressure at the input of these two major vessels. 
These curves may be broken in to their respec- 
tive Fourier series. Each term in the series may 
be represented by a complex number. The ratios 
of the corresponding harmonics of pressure and 
flow permit the computation of the complex im- 
pedance. The results to date indicate that the 
input impedance to the pulmonary artery con- 
sists of a very large real term and a relatively 
small imaginary term. Although the interpreta- 
tion of this finding is somewhat involved, the 
major impedance to blood flow into the pulmo- 
nary artery seems to be related to vascular re- 
sistance. This is in contrast to the systemic side 
in which large reactive components of impedance 
appear. 

Pulmonary Biophysics. Theoretical considera- 
tions have been developed which indicate that 
abnormal stress distribution within the structure 
of the lung may be the major factor in the pro- 
duction of the disruptive lesions of emphysema. 
The abnormal stress distribution could be the re- 
sult of either congenital or acquired abnormali- 
ties of the smaller terminal air passages. There- 
fore, studies have been undertaken to examine the 
relationship of stress to strain in three dimensions 
in excised lungs. By using an improved intra- 
esophageal pressure measuring system, the intra- 
thoracic pressure is being measured simultane- 
ously at three different sites along the esophagus. 
The difference between oral pressure and the in- 
traesophageal pressures has been studied under 
static conditions in normal subjects. It has been 
possible to describe this relationship as a func- 
tion of balloon volume, balloon position, and lung 
volume. The relationship seems to be independ- 
ent of the rate of change of pressure or of the 
amplitude of pressure changes. This initial part 
of the project, which is of interest in itself, is 
preliminary to studying the pressure differences 
between the three balloons and the conditions of 
air flow, varying rates of respiration, and rapid 
respiratory maneuvers such as cough. If it can 
be demonstrated that there are significant differ- 
ences in the stress distribution in the lung of nor- 
mal individuals, as compared to those with 
chronic bronchitis, evidence is gained for support 
of the theory that the disruptive lesions of pul- 



106 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



monary emphysema may be the result of abnor- 
mal physical stress. 

New methods of studying the mechanical be- 
havior of the lung have been developed. A unified 
three dimensional presentation of the three major 
variables controlling the lung behavior in health 
and disease has been developed in this laboratory 
and used to examine normal, cardiac, and em- 
physematous subjects. From these considerations 
a unique relationship between the maximum 
achievable expiratory flow and the degree of lung 
inflation has been discovered. Theoretical con- 
siderations of this unique flow-volume relation- 
ship have far reaching physical and physiological 
implications. Therefore, this relationship is be- 
ing extensively studied in normal human beings. 
Theory would indicate that this relationship is 
controlled by the density and viscosity of the 
gas as well as the dimensions and physical prop- 
erties of the intrathoracic airways. If it can be 
shown, experimentally, that the effect of either 
density or viscosity on this relationship was of 
minor importance, it would greatly simplify the 
mathematical description of this curve. Prelimi- 
nary findings indicate that the viscosity of the 
gas breathed has very little effect on the maxi- 
mum achievable expiratory flow at any given 
degree of lung inflation. This finding is in sharp 
contrast to the effect of gas density. If these 
preliminary findings are verified, it may be pos- 
sible to establish a realistic and useful mathe- 
matical model of the lung from which the flow 
volume curve may be analyzed mathematically. 



SURGERY BRANCH 

The investigative projects of the Section on 
Clinical and Experimental Surgery of the Sur- 
gery Branch have, as in past years, centered 
largely around methods for improving the surgi- 
cal treatment of patients with congenital or ac- 
quired heart disease and in elucidating the 
physiologic factors which apply before, during 
and following the operative correction of such 
lesions. An important group of studies has con- 
tinued to center around the clinical application 
of the artificial heart and lung machine. The 
artificial heart and lung machine itself has not 
been modified in the past year since it had been 
found to provide adequate support of the circula- 



tion for periods of up to two hours. It may be 
of interest that the machine has been accepted 
for commercial production. 

Perhaps the most pressing clinical problem in 
the field of cardiovascular surgery is the surgical 
correction of mitral and aortic regurgitation. 
For the past two years, efforts in both the experi- 
mental laboratory and in the operating room have 
been directed at the perfection of a prosthesis 
suitable for total replacement of the mitral valve. 
Valves have been constructed with a dacron fiber 
base covered with a thin layer of polyurethane 
foam. They have been implanted into five pa- 
tients with irretrievably damaged valves. Al- 
though no patient is presently living with a total 
valve replacement, the valve was in place in one 
patient for more than four months. Autopsy 
examination revealed that this valve was free of 
clot, covered with a thin layer of fibrin and had 
remained mobile. This result is sufficiently en- 
couraging that further refinements in the design 
of the valve are being made in the hope that it 
will ultimately prove useful for routine clinical 
practice. An important part of the problem of 
prosthetic valve replacement, whether for the 
aortic or the mitral valve, is a choice of material. 
It is particularly difficult to evaluate materials 
when valves are constructed and placed in ex- 
perimental animals since the technical aspects of 
the operation itself are so formidable. Accord- 
ingly, various fabrics such as dacron, silk, silastic, 
pericardium and nylon have been implanted into 
the wall of the right atrium in dogs. A study 
of the behavior of these materials in this loca- 
tion will undoubtedly indicate that certain fab- 
rics are more desirable than others in valve con- 
struction. 

In the prosthetic mitral valve which has been 
employed clinically, artificial chordae tendinae 
are necessary to prevent eversion of the valve 
leaflets from the ventricle into the atrium. An 
experimental study is underway to evaluate the 
ideal material for such artificial tendinae. Strands 
of various materials, such as stainless steel, silk, 
silk covered with plastic, etc., have been passed 
through the substance of the heart so that the 
fibers traverse the cavity of each ventricle. In 
addition, various methods have been employed 
to fix the strands to each ventricular wall. Pre- 
liminary observations indicate that clotting is 
primarily related to the point at which the 



NATIONAL HEART INSTITUTE 



107 



strand pierces the endocardium and that the sub- 
stance of the strand may be of lesser importance. 
Fixation of the strands by mechanical means has 
been found inferior to direct suture. 

In the clinical treatment of patients with vari- 
ous types of congenital and acquired heart dis- 
ease principal efforts in the past year have been 
directed at surgery of the mitral and aortic 
valves. Continuing experience with the open 
surgical correction of calcific aortic stenosis, with 
followup catheterization information has revealed 
that in the majority of patients simple commis- 
surotomy is not effective in relieving left ven- 
tricular outflow obstruction unless aortic regurgi- 
tation is produced. Accordingly, a portion of the 
aortic valve has been replaced with a prosthesis, 
ordinarily of woven teflon cloth, in an increasing 
number of patients. It soon became apparent 
that the prolonged period of aortotomy necessary 
in these operations could not be provided by 
coronary perfusion alone. For this reason selec- 
tive hypothermia of the heart, induced by perfu- 
sion of the coronary vessels with refrigerated 
blood and a slush of ice and water around the 
left ventricle has been adopted. Utilizing this 
technique, safe periods of cardiac arrest up to 
two hours have been achieved. A study com- 
paring the effects of cardiac hypothermia and 
coronary perfusion on ventricular function has 
been initiated. 

One year ago two patients with functional 
hypertrophic subaortic stenosis were subjected to 
operation. It is impossible to resect the obstruct- 
ing muscle mass and in these patients the lesion 
was treated by a vertical incision or myotomy 
across the contraction ring within the left ven- 
tricle. Although the initial hemodynamic results 
seemed encouraging, the procedure was not again 
used until these patients were studied one year 
later. At this time each was found to have virtu- 
ally complete relief of the obstruction. Since 
increasing numbers of patients with this form 
of aortic stenosis are being seen, it is likely that 
this new operation will prove of increasing value. 

Previous studies have indicated that it is ad- 
visable for every patient undergoing open heart 
surgery to be digitalized beforehand. There has 
been much conjecture as to whether or not extra- 
corporeal circulation removes digitalis from the 
heart muscle and whether patients after open 
operations should receive additional digitalis. An 



experimental study was undertaken in which ani- 
mals were digitalized and their digitalis level 
titrated before and after cardiopulmonary by- 
pass. Preliminary work indicates that digitalis 
is fixed in heart muscle and no decrease in its 
effective level could be demonstrated after bypass. 
The work will be continued, more precisely, with 
the use of radioactive digitalis preparations. 

It has been occasionally noted in patients un- 
dergoing prolonged cardiopulmonary bypass that 
oliguria or anuria occurs. Although a high level 
of free plasma hemoglobin is frequently present 
following long perfusions there has been no 
correlation between the occurrence of a high 
plasma hemoglobin level and the occurrence of 
renal complications. In an experimental study, 
p-aminohippurate and creatinine are being util- 
ized to measure renal plasma flow and glomeru- 
lar filtration rate in dogs in which the kidney is 
supported by an extracorporeal circulation. By 
means of this preparation the effects of pure 
hemoglobin and red cells debris on renal func- 
tion may be evaluated. 

In many clinics extracorporeal circulation is 
presently being used concurrently with deep hy- 
pothermia, heat exchange being accomplished in 
the extracorporeal circuit. It has been gener- 
ally assumed that as the body temperature falls 
blood flow to muscle masses is decreased while 
that to the "vital organs" remains high or is in- 
creased. Experimental studies have been under- 
taken to measure the total blood flow and the 
vascular resistance of various circulatory beds 
during profound hypothermia. It has been a 
surprise to learn that at 10°C. the flow to the 
lower extremity is increased while that to the 
brain and splanchnic bed is lower than normal. 
Continuing investigations concerning the changes 
in hepatic blood flow under these circumstances 
are underway. When profound hypothermia is 
used, ventricular fibrillation is an almost invari- 
able complication of the technique and in clinical 
practice large doses of quinidine and sometimes 
procaine amide have been given to prevent this 
arrhythmia. The direct myocardial effects of 
these agents were not known and, accordingly, 
left ventricular function curves were constructed 
in dogs in which quinidine had or had not been 
given. It was found that quinidine produced a 
marked depression of left ventricular function 
and a profound fall in mean arterial pressure 



108 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



indicative of peripheral vasodilation. It would 
seem that the protective effects of quinidine are 
outweighed by these undesirable side effects. 

Fifteen patients have now been studied in 
whom left ventricular outflow obstruction was 
found to be due to massive hypertrophy in the 
outflow tract of the left ventricle. An attempt 
to produce this lesion experimentally was made 
by constriction of the ascending aorta in pup- 
pies. These animals have now been followed for 
one year and all have been found to have massive 
hypertrophy of the outflow tract of the ventricle 
and in several a pressure gradient within the 
left ventricular cavity has been found at retro- 
grade arterial catheterization. Although this ex- 
perimentally produced lesion is probably not akin 
to the asymmetric hypertrophy most common in 
patients, it does provide an experimental tool by 
which operative treatment of this unusual form 
of aortic stenosis may be evaluated. 

The complete anatomic correction of certain 
congenital lesions such as severe tetralogy of Fal- 
lot and true truncus arteriosus will necessitate 
replacement of the pulmonary artery. In a pre- 
vious study, single pulmonary arteries were re- 
placed with grafts of plastic material and this 
work has been extended to permit total replace- 
ment of the main pulmonary artery and its major 
branches. It has been found that the length of 
the rigid graft is of critical importance but that 
when this parameter is controlled, that dogs can 
survive for prolonged periods with such a pros- 
thesis not containing a valve. 

While the heart and peripheral circulation are 
physiologically separated during cardiopulmon- 
ary bypass, a unique opportunity is afforded for 
studying the various direct myocardial and peri- 
pheral actions of many pharmacologic agents. 
The effects of various pressor amines and digi- 
talis have been described and these studies sug- 
gested that a detailed investigation of certain 
anesthetic agents would be of value. In a study 
carried out in conjunction with the Department 
of Anesthesiology the anesthetic agent Halothane 
was introduced into the oxygenator for a period 
of five minutes. In six patients it was shown 
that there was immediate and progressive depres- 
sion of myocardial contractility and a concomi- 
tant fall in systemic blood pressure indicating 
peripheral vasodilation. The effects of carbon 
dioxide and other gaseous agents on the heart 



and peripheral circulation can also be studied by 
this method and such projects have been ini- 
tiated. The clinical observation was made that, 
following operations necessitating occlusion of 
the thoracic aorta, paradoxical hypertension fol- 
lowing restoration of the circulation was far 
more frequent when Halothane anesthesia was 
employed. In an experimental preparation the 
aorta was occluded in dogs under either nitrous 
oxide-oxygen or Halothane anesthesia. Postop- 
erative hypertension was uniformly noted when 
Halothane was employed. This action of Halo- 
thane may originate in an alteration of renal 
blood flow and this aspect of the problem is under 
investigation. 

During the past year the Surgery Branch has 
supported a Resident in Orthopedic Surgery. 
Two investigative projects have been carried out 
in collaboration with the National Institute of 
Arthritis and Metabolic Diseases. There is no 
detailed knowledge concerning the pathogenesis 
of the bone changes secondary to cyanotic heart 
diseases. Various experimental procedures have 
been employed to reduce arterial oxygen satura- 
tion and a series of animals in which cyanosis 
has been produced are being X-rayed at inter- 
vals to determine the progression of their pul- 
monary osteoarthropathy. Also a new lesion of 
the small blood vessels in joint tissue has been 
observed in a series of patients who had no 
symptoms of joint disease. The lesion can be 
characterized as an occlusive vascular one whose 
significance or etiology is entirely unknown. A 
detailed investigation of the incidence of this 
vascular lesion has been undertaken in an effort 
to determine its possible relationship to the aging 
process. 

Clinical studies in the Section of Cardiology 
during the past year have again focused on the 
applicability of Starling's law of the heart to 
man. A method was developed by which it is 
possible to determine directly the interrelation- 
ships between ventricular end-diastolic fiber 
length, diastolic tension, systolic tension and the 
rate of development of tension. These measure- 
ments may all be carried out by means of a spe- 
cifically modified Walton-Brodie strain gauge 
arch sewn to the surface of the human heart. 
These studies have shown that as human myo- 
cardial fibers are progressively stretched, a pro- 
gressive increase in diastolic tension, in the ten- 



NATIONAL HEART INSTITUTE 



109 



sion developed during systole, and in the rate of 
development of tension takes place. These ob- 
servations are direct evidence of the operation 
of Starling's law of the heart in man. In other 
studies, on patients with mitral stenosis and atrial 
fibrillation, further evidence of the applicability 
of Starling's law was obtained. Continuous al- 
terations of the length of a segment of left ven- 
tricular muscle were recorded at operation. On 
a beat-to-beat basis the length of a segment of 
left ventricular muscle at the end of diastole was 
found to correlate extremely well with the char- 
acteristics of ventricular contraction; the latter 
was assessed by a detailed analysis of individual 
left ventricular pressure pulses. Similarly, the 
left ventricular end-diastolic pressure appeared 
intimately related to the subsequent ventricular 
contraction in several patients with a closed chest 
studied at the time of left heart catheterization. 
Ventricular function or "modified Starling" 
curves were obtained in seven subjects with nor- 
mal cardiovascular systems studied while under 
complete ganglionic blockade. "Effective" left 
ventricular end-diastolic pressures, cardiac out- 
put, and arterial pressure before and after the 
transfusion of 1,500 ml. of blood were measured. 
In each instance ventricular filling pressure cor- 
related well with the left ventricular stroke vol- 
ume and stroke work, further supporting the con- 
cept that Starling's law of the heart applies to 
man. 

Hemodynamic observations on the function of 
the left atrium were carried out on 50 patients 
with various forms of heart disease. It was 
found that in patients with left ventricular dis- 
ease (arteriosclerosis, aortic valve disease, etc.) 
left atrial contraction elevated left ventricular 
end-diastolic pressure while maintaining the left 
atrial (and therefore the pulmonary capillary) 
pressure at a substantially lower level. This dis- 
sociation between left ventricular end-diastolic 
and mean left atrial pressures provides an "in- 
dex" of left atrial function and emphasizes the 
clinical importance of left atrial contraction in 
patients with left ventricular hypertrophy. It 
was shown that, just as in the left ventricle, the 
pressure in the left atrium just prior to the onset 
of atrial contraction is an important determi- 
nant of the characteristics of atrial contraction. 

Currently, measurements of two other basic 
parameters are being carried out. The left ven- 



tricular end-diastolic volume is being correlated 
with the left ventricular stroke volume and 
stroke work. Similarly, the rate of development 
of left ventricular pressure, i.e. the slope of the 
isometric pressure gradient, is being correlated 
with the end-diastolic segment length. These 
studies are designed to provide more detailed in- 
formation about the hemodynamic determinants 
of the characteristics of ventricular contraction 
in man. 

In a continuing investigation of the pharma- 
cology of drugs which act primarily on the car- 
diovascular system it was shown that digitalis 
glycosides augment the contractile force of non- 
failing human hearts. In these studies, carried 
out on patients at the time of "open heart" op- 
erations it was also observed that digitalis exerts 
a direct arteriolar constrictor effect. These 
studies may serve to change current concepts of 
"prophylactic digitalization." In other observa- 
tions on digitalis glycosides it was demonstrated 
that the production of mild hyperthyroidism 
greatly augments digitalis requirements. It was 
shown that large doses of rauwolfia alkaloids 
abolish these increased digitalis requirements. 
Guanethidine, a drug which apparently releases 
tissue stores of catecholamines, was found to 
abolish many of the manifestations of triiodothy- 
ronine-induced hyperthyroidism. In other studies 
the effect of large doses of syrosingopine, a rau- 
wolfia derivative, on the cardiac response to acute 
hypoxemia and exercise was evaluated. It was 
found that the tissue catecholamine depletion pro- 
duced by this drug failed to alter the cardio- 
vascular response to these two stimuli. 

The development and evaluation of newer diag- 
nostic technics was continued during the past 
year. In a critical appraisal of the Kr 85 inhala- 
tion test for the detection of left-to-right circu- 
latory shunts, in over 300 patients in whom the 
presence or absence of such a shunt was subse- 
quently proved, it was shown that the Kr 85 in- 
halation test probably represents the most accu- 
rate means for the clinical detection of circulatory 
shunts. A simplified technique for the detection 
of patent ductus arteriosus was developed. This 
consists of the injection of Kr 85 into the thoracic 
aorta and measuring the appearance time in the 
expired air. This technique has been found par- 
ticularly valuable in excluding a patent ductus 
arteriosus in patients prior to open heart opera- 



110 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tions. Both ascorbic acid and iced saline have 
also been employed as indicators in the study of 
patients with congenital heart disease. Ascorbic 
acid is detected by means of a platinum electrode, 
while temperature is sensed with a small thermis- 
tor. Both of these elements may be introduced 
directly into the arterial blood or into the heart. 
By these techniques indicator-dilution curves may 
be recorded without sampling blood and there- 
fore they constitute an important advance in the 
use of indicator-dilution curves. Solutions of 
Kr 85 have also been employed as the indicator for 
the measurement of cardiac output by the indi- 
cator-dilution technic. Since Kr 85 does not recir- 
culate, inscription of the dilution curve is obvi- 
ated, thus greatly simplifying the technic of 
cardiac output measurement. Transseptal left 
heart catheterization, developed in this laboratory 
2 years ago, has been modified so that : (1) surgi- 
cal exposure of the saphenous vein is avoided, 
(2) a smaller needle is employed for atrial punc- 
ture, (3) a larger catheter can be introduced into 
the left side of the heart, and (4) left heart 
angiography may be performed conveniently. 
This has greatly improved the clinical value of 
this technic. 

Observations on 15 patients with idiopathic 
hypertrophic subaortic stenosis have been con- 
tinued. This is the largest number of patients 
studied at any single hospital and detailed clini- 
cal, phonocardiographic, hemodynamic and angio- 
graphic observations have permitted a compre- 
hensive description of this disease entity. In 
particular, a simple hemodynamic technic for the 
detection of this condition was described. This 
technic is based on an analysis of the response 
of the arterial pressure pulse to a premature ven- 
tricular contraction. 

In studies performed in the experimental labo- 
ratory of the Section of Cardiology the effects 
of a variety of stimuli on the arteriolar and 
venous tone of the dog were examined. These 
investigations were carried out employing a prep- 
aration on total cardiopulmonary bypass in which 
the heart and lungs were excluded from the cir- 
culation. Thus, it was shown that veno-constric- 
tion accompanies arterial constriction when the 
pressure in the carotid sinuses and in the left 
side of the heart are lowered. Similarly, hypoxia 
and hypercapnia produce veno-constriction, but 
the increased arterial pressure which usually oc- 



curs with these stimuli results from an accom- 
panying increase in the cardiac output. Hypo- 
thermia was shown to produce arterial and venous 
dilatation while hyperthermia has the opposite 
effect. 

The mechanism of the inotropic, chronotropic 
and pressor effects of guanethidine was studied 
in normal dogs and in dogs following chronic 
cardiac denervation. The results of these experi- 
ments support the contention that guanethidine 
produces a positive inotropic and chronotropic 
effect by suddenly releasing the myocardial stores 
of catecholamines. 



GERONTOLOGY BRANCH 

The resarch program of the Gerontology 
Branch is directed toward (1) describing the 
biochemical, physiological and psychological 
changes that take place with increasing age in 
man and (2) investigating the basic biology of 
aging in order to understand age-dependent alter- 
ations in performance in man. 



Physiological Studies 

Longitudinal Studies 

Age differences in biochemical, physiological 
and psychological characteristics of normal peo- 
ple still living successfully in the community are 
being evaluated. These subjects, ranging in age 
from 18 to 100 years, have agreed to return to 
the laboratory every 18 months for the remainder 
of their lives so that age changes can be recorded 
in individual subjects. Almost 200 subjects have 
received the first series of tests and 50 have been 
tested a second time. The program has been well 
received as evidenced by the fact that we are now 
making appointments with new recruits for De- 
cember 1961. Due to limitations in space and 
staff, we can accept only two new subjects per 
week. 

Because of the long term nature of this project, 
results on successive measurements cannot be 
analyzed as yet. However, members of this lon- 
gitudinal sample have served as experimental 
subjects for many of the other studies on humans. 

In collaboration with Dr. Bernice Cohen of the 
Johns Hopkins School of Hygiene and Public 



NATIONAL HEAET INSTITUTE 



111 



Health, the testing schedule has been expanded to 
include information on genetic background which 
should shed light on the question of the relation 
of assortive mating to longevity. Dietary habits 
of the subjects will also be surveyed in collabora- 
tion with the Maryland State Health Department 
and the Heart Disease Control Program of the 
USPHS (Dr. McGandy). 

Renal Studies 

Experiments have been conducted to determine 
whether the hemoglobin-haptoglobin complex is 
cleared from the plasma by the reticulo-endo- 
thelial system. Preliminary experiments indicate 
that Kupffer cells may be isolated from liver 
homogenates. If this method is successful, ex- 
periments will be performed using hemoglobin 
tagged with Fe 59 to determine the presence of 
the hemoglobin-haptoglobin complex in isolated 
Kupffer cells in the dog. 

No age differences in glomerular permeability 
were found using infusions of dextran of differ- 
ent molecular weights. 

Studies of glomerular filtration of free hemo- 
globin (corrected for binding to haptoglobin) 
relative to inulin clearance have been carried out 
in 47 subjects aged 20-88 years. The ratio Chi>/ 
Cm averaged 0.055 (S.D. 0.019) and showed no 
significant correlation with age. Thus there is 
no evidence of a change in glomerular perme- 
ability with age. Tubular reabsorption of hemo- 
globin, calculated as the difference between fil- 
tered load and urinary excretion averaged 1.43 
mg./min (S.D. 0.96). 

Measurements of creatinine clearance made over 
a period of 12 hours indicate similar rates of 
decline with age in both hospitalized and com- 
munity residing groups. 

Body Composition Studies 

Estimates of lean body mass and body fat 
have been made utilizing the equations proposed 
by Keys and Siri which are based on body den- 
sity and total body water determinations. In 
addition, a new equation was developed which 
introduces corrections for bone density (estimated 
from X-ray measurements of the phalanx) and 
for muscle mass (estimated from creatinine ex- 
cretion) . "When fat content of the body was cal- 
culated from the Keys equation and expressed as 
a percentage of body weight an increase of 0.06 



percent per year was found. However, when the 
new equation was used there was a decrease of 
0.12 percent per year. The new equation gives 
a better estimate of body fat by introducing cor- 
rections for bone density and muscle mass, both 
of which show significant age decrements. 

Energetics of Arm Motion 

In cooperation with Dr. R. L. Ramsey, Medi- 
cal College of Virginia, a description of the me- 
chanical relationships of the human arm swinging 
maximally in alternating back and forth move- 
ments has been developed. During this maximum 
wagging exercise linear relationships were found 
between (1) period of swing and angular dis- 
placement (amplitude), (2) the impulse of the 
force applied to the limb and the log of the 
angular displacement and (3) action and angular 
displacement. The hypothesis that the slopes of 
these relationships were related to mechanical 
efficiency was not supported for any of them. 
Mechanical efficiency was calculated from the ex- 
cess oxygen consumption associated with the work 
done. A family of curves was developed which 
related mechanical efficiency to age at different 
amplitudes of swing. Young subjects (age 30) 
showed an increase in efficiency with increasing 
amplitude of swing, whereas in old subjects (age 
70) efficiency did not increase with increasing 
amplitudes of swing. In both old and young sub- 
jects, the angular proportion of the swing during 
which force was exerted by the muscle decreased 
(93 percent to 57 percent), with increase in am- 
plitude (0.36 Rad to 1.85 Rad). 

The delineation of the mechanics of arm move- 
ment provides a basis for understanding age 
changes in the ability of an individual to perform 
simple tasks efficiency. These measurements may 
provide a simple objective test of muscular effi- 
ciency that can serve as one factor in an index 
of aging. 

Psychological Studies 

Investigators in the Psychology Section provide 
information on the psychological performance 
and personality characteristics of subjects in- 
cluded in the longitudinal study of aging. In 
addition to standard tests of intellectual per- 
formance, standard questionnaires to evaluate 
personality characteristics are being administered. 



112 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Estimates of reaction time, a-frequency of the 
EEG, spinal reflex amplitude, heart rate varia- 
bility and motor time in these subjects are also 
being made. 

A substantial correlation (0.81 with p <0.01) 
between mean reaction time and mean alpha wave 
frequency of the electroencephalogram has been 
observed in a group of 13 subjects. Correlation 
between these variables within individuals varied 
over a wide range of values (+0.03 to +0.65). 
When the effects of several other variables re- 
lated to reaction time were partialed out by means 
of multiple regression analyses, the low individual 
correlation of 0.03 between reaction time and 
alpha wave frequency was increased to 0.29. The 
variables partialed out were amplitude of spinal 
reflex, heart rate and motor time (the time which 
elapses between the appearance of a muscle action 
potential and the mechanical response of the 
muscle) . These variables are regarded as indices 
of the activity level of the brain stem reticular 
system. These measures also account for y s of 
the total variance of the speed of response. Hence, 
it may be inferred that speed of response is in- 
fluenced by general excitatory states of activity 
in the brain and spinal cord which may fluctuate 
from moment to moment. 

The effect . of interpolated activities on short 
term memory was tested in subjects with a high 
level of educational attainment. In contrast to 
the previous study which showed greater inter- 
ference among old than young subjects with a low 
level of education, age differences in this group 
were not statistically significant. However, when 
the task was shifted to the judgment of a time 
interval (length of time a stimulus light was on) , 
the older subjects showed a significantly greater 
interference effect. In this experiment the young 
subjects were more effective than the old in learn- 
ing the original stimulus series so that a covari- 
ance analysis was required to test the significance 
of the age difference in re-learning scores. This 
analysis showed that the age difference was sta- 
tistically significant. It is inferred that external 
cues were available in the experiment requiring 
judgments of size which were not available in 
the experiment involving time judgments and 
that the group of more capable subjects used 
these cues to a greater extent than did the sub- 
jects of lower capabilities. 



Basic Biology 

Cellular and Comparative Physiology 

The general research objectives and activities 
of the Cellular and Comparative Physiology Sec- 
tion continue in a twofold direction: (1) the de- 
scription of cellular and organismic changes in 
humans and appropriate experimental animals 
during aging and (2) the measurement of the 
effects of alterations in the environment on the 
performance and mortality of experimental ani- 
mals. 

Histochemical methods for the localization of 
reductases in tissue cultures of developing em- 
bryonic muscle tissue have made possible the dem- 
onstration that succinic dehydrogenase, DPN • 
H-cytochrome c reductase and TPN • H-cyto- 
chrome c reductase increase dramatically con- 
comitantly with multinuclearity. This finding 
indicates an increase in mitochondria following 
cell fusion. 

In order to determine whether the multinuclear 
cell becomes dependent on oxidative pathways 
exclusively, cultures were grown in the presence 
of antimycin A. The results indicate that al- 
though the mononucleated cell can maintain itself 
and proliferate in the presence of the blocking 
agent, the multinucleated cells are selectively 
killed. Thus muscle differentiation may depend 
upon an obligatory aerobic metabolic process. 
These studies lend support to the hypothesis that 
growth (cell proliferation) and differentiation 
are mutually exclusive processes. 

Histochemical localization of reduced nucleo- 
tide reductases has also been useful in demon- 
strating cross-striations in developing muscle tis- 
sue in tissue cultures. These striations can be 
detected since the mitochondria are oriented along 
the A bands. 

The recording spectrophotometer has been 
modified to permit continuous scanning of the 
absorption spectrum of individual cellular ele- 
ments. By using appropriate corrections, the 
standard error of replicate readings has been re- 
duced to 1-3 percent of the mean value. 

Using this method, measurements of ploidy 
variability in developing chick muscle cultures 
have been made to compare the distributions in 
mononuclear and multinuclear cells. Preliminary 
results show a unimodal distribution of DNA 
values for the muscle cell nuclei which corre- 



NATIONAL HEART INSTITUTE 



113 



sponds to the lower peak (diploid) of the bimodal 
distribution found in mononuclear cells. If this 
result is confirmed, it will offer evidence that 
muscle is a true syncytium. This technique will 
make it possible to determine whether the nuclei 
of the multinuclear muscle cells are, in fact, non- 
proliferative. Measurements of DNA per cell 
will be extended to comparisons of cells from 
young and old animals to test the hypothesis that 
the frequency of mitotic "accidents" increases 
with age. 

Muscle cells obtained from biopsy material 
taken from adult and aged humans can be main- 
tained in tissue culture. Cells originating from 
mature skeletal muscle tissue are capable of neo- 
formation of multinuclear muscle cells. After 
2—3 weeks of culture, long multinuclear ribbons 
form in the outgrowth area of the original ex- 
plants. These resemble the early multinuclear 
cells of chick embryonic muscle and have a highly 
refractile cytoplasm which, after fixation and 
staining, shows many longitudinal fibrils. Re- 
moval of the original explants prior to multi- 
nuclear cell formation did not affect the process. 
This indicates that the multinuclear cells do not 
arise simply by the "budding" of injured mature 
muscle cells. Efforts are being made to isolate 
the various cell types found in mature muscle in 
order to determine the source of the myogenic 
elements. 

Further studies have been made of the age pig- 
ments which accumulate in the human myocar- 
dium. The particles have the following average 
dimensions: major axis, 1.39 ± 0.06 micra; minor 
axis, 0.97 ± 0.04 micra. Specific gravity of the 
particle is 1.18 ± 0.03. Measurements of reduced 
vs. oxidized difference spectra indicate that iso- 
lated age pigment particles contains very little 
flavoprotein, cytochrome or cytochrome c reduc- 
tase which are present in mitochondria. Tests for 
some of the Krebs cycle oxidases have also been 
negative. Cathepsin and acid phosphatase activi- 
ties were pronounced and there were traces of acid 
deoxyribonuclease and ribonuclease in the par- 
ticles. Thus the enzymatic behavior of the age 
pigment particles resembles that of lysozymes 
more than mitochondria. 

Alkaline hydrolysis of lipids extracted from 
age pigment does not greatly alter the fluores- 
cence. Gas chromatography of fluorescent frac- 
tions separated on silicic acid columns and 



subjected to methanolysis has revealed a prepon- 
derance of unsaturated over saturated fatty acid 
esters. The yellow fluorescent material also shows 
an infrared spectrum very similar to that of old 
oxidized samples of animal cephalin. 

Data have been obtained that indicate that the 
photoreduction of TPN (in spinach chloroplasts) 
does not require the utilization of high energy 
phosphate bond energy (ATP) . The firefly assay 
system was adapted for the continuous measure- 
ment of ATP levels in illuminated chloroplast 
suspensions. 

Studies of the effects of environmental factors 
on the longevity of Drosophila melanogaster have 
been continued. Flies reared in environments 
free from micro-organisms live slightly longer 
than flies reared in a nonsterile environment. 
Exposure to 50,000 B, of radiation increases (by 
50 percent) the longevity of flies in nonsterile 
environments, but reduces longevity by about 20 
percent in flies reared under sterile conditions. 
When flies are reared in an environment of 100 
percent oxygen, the rate of aging is increased by 
about 50 percent, whereas, at lowered oxygen 
tensions (1-2 percent O2), the rate of aging is 
reduced by about 35 percent. 

Extensive time lapse records of the develop- 
ment, function, senescence and death of a number 
of species of coelenterates have been made. The 
individual species of this phylum have widely 
different longevities, ranging from a few days to 
many years. Stolonic fusion, hydranth regres- 
sion, digestion, respiratory movements, growth, 
cleavage, and other functions have been recorded. 
Senescence and death of individual hydranths of 
Campanularia begins with a gradual contraction 
of the tentacles and is followed shortly by lysis 
and contraction of the hydranth with the return 
of its contents to the colony. 

Nutritional Biochemistry 

Three general areas have been investigated 
during the past year within the Section on Nutri- 
tional Biochemistry. These included studies on 
(1) age differences in tissue metabolism of the 
rat, (2) biochemical and physiological differences 
between normal rats and those whose life span 
may be increased through dietary restriction and 
(3) the effect of diets with respect to their poten- 
tial to increase life span. 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Among these studies has been an investigation 
on the effect of age on protein catabolism. Pre- 
vious studies have indicated a high catheptic 
activity in the livers and kidneys of senescent 
rats. Since the in vitro determination of the en- 
zymatic activity measures the breakdown of pro- 
tein, a higher rate of catabolism of tissue proteins 
in old animals may be inferred. The earlier study 
(age differences in enzymatic activities of tissues 
during protein depletion and repletion) may be 
criticized on the grounds that it did not measure 
protein metabolism in normal animals. There- 
fore, the rate of protein catabolism was estimated 
by injecting radioactive methionine into animals 
of different ages and measuring the rate of dis- 
appearance of S 35 from the soluble protein frac- 
tions of various tissues. Although the rate of 
loss of radioactivity in the soluble protein frac- 
tion of liver tended to be higher in the older 
animals, statistical significance could not be estab- 
lished in this tissue nor in kidney, heart or muscle. 
Thus these studies again have failed to demon- 
strate any effect of age on protein metabolism. 

Mitochondria from liver and kidney tissues of 
old and young rats have been separated. In both 
tissues succinoxidase activity per unit of washed 
mitochondria was the same for both old and 
young rats. The evidence thus suggests a senes- 
cent loss of whole mitochondria rather than a 
gradual loss of enzyme per mitochondrion. Tn 
these experiments, protein nitrogen content and 
turbidity of washed suspensions were used to esti- 
mate the number of mitochondria in the suspen- 
sions. The use of millipore filters for separating 
mitochondria is being explored to facilitate actual 
counting of the mitochondria. Studies are also 
being conducted to produce suspensions of isolated 
cells from other tissues such as the central nervous 
system for use in metabolic studies. Preliminary 
experiments show that incubation of minced cere- 
bellum with papain and versene yields a suspen- 
sion containing free Purkinje cells which can 
probably be isolated for metabolic studies and 
the preparation of mitochondria. 

In past experiments, the age differences demon- 
strated in the concentrations of various enzymes 
in the kidney tissue of the rat have been rela- 
tively small (10 percent) in comparison to the 
large decrements in renal function observed in 
human subjects (60 percent). It is possible that 
aging affects the kidneys of these two species at 



different rates or that the enzymes chosen for 
investigation were not sensitive indices of renal 
function. Therefore, attempts were made to de- 
termine the effect of age on renal tubular trans- 
port in an in vitro system by measuring the ac- 
cumulation of PAH by renal cortical slices from 
rats. Tubular function was estimated by the 
ratio of the concentration of PAH in the slice 
to that in the medium (S/M). The results dem- 
onstrated a tendency toward slightly lower S/M 
values in the senescent animals, but even this 
decrement was explained by a decrease in the 
number of cells as measured by the concentration 
of DNA in the tissue samples. 

In another study, an estimate of age differences 
in total renal capacity was measured by the 
amount of renal tissue produced following uni- 
lateral nephrectomy in the rat. In addition to 
the total amount of tissue regenerated, the con- 
centrations of succinoxidase, alkaline phosphatase, 
DNA, UNA and protein nitrogen were also de- 
termined. Although a greater degree of hyper- 
trophy was found in the young animals (44 per- 
cent) as compared to senescent ones (33 percent), 
the concentrations of the various tissue compo- 
nents (DNA, UNA, protein N and enzymes) per 
unit wet weight in the hypertrophied kidneys did 
not vary by more than 10 percent from values 
found in the normal kidneys and no marked age 
differences were observed. At the present time, it 
is not possible to attribute the observed differ- 
ences in the degree of hypertrophy specifically to 
aging since the older animals, as indicated by 
body weight changes, did not seem to recover 
from the operation as well as the young. 

In view of the fact that the same tests have 
not been carried out in the rat as in man, clear- 
ance techniques for the estimation of renal func- 
tion in the rat are being developed. At present 
it is possible to measure renal clearances in un- 
anesthetized rats under conditions in which inu- 
lin and PAH plasma levels remain constant for 
thirty minutes and the bladder is adequately 
rinsed to assure complete urine collecting. In 
future experiments, it will be possible to compare 
the effect of age in man and the rat by the same 
renal function tests. 

For many years, it has been well known that 
the life span of the rat and mouse can be in- 
creased by severe dietary restriction. In order 
to understand the mechanism of this phenomenon, 



NATIONAL HEART INSTITUTE 



115 



experiments were carried out to determine the 
biochemical and physiological differences between 
normal animals and those subjected to such die- 
tary restriction. The results of this experiment 
demonstrated increased concentrations of succin- 
oxidase in liver tissue and alkaline phosphatase 
in kidney tissue of the restricted animals. Since 
the previous study had indicated that the concen- 
trations of these enzymes are lower in younger 
animals, these data do not support the concept 
that the restricted animal is similar to a chrono- 
logically younger animal. Similarly, the activity 
patterns measured in both suspension and wheel 
type cages also fail to support the hypothesis 
that the restricted animal is similar to a chrono- 
logically younger animal. Although dietary re- 
striction has been shown to increase the life span 
in rats and mice, the application of this principle 
to species exposed to everyday stress is questioned 
in view of the work of McCay showing that the 
restricted dog succumbs much more readily to 
bacterial infection and parasitic infestation than 
does the normal animal. Since our experiments 
demonstrated that alterations in the concentra- 
tions of certain tissue enzymes do occur during 
dietary restriction, it may be possible to choose 
a diet which will support normal growth and still 
provide the necessary enzymatic alterations in the 
tissues to increase life span. Our first experiment 
in this direction demonstrated that it is possible 
to increase the concentrations of selected enzymes 
in various tissues by feeding growing animals 
diets which will promote optimum growth but 
which differ only in the quality (casein vs. whole 
dessicated liver) or quantity (20 to 32 percent 
protein) of the dietary protein. Thus far, these 
experiments have not been totally successful since 
the enzymes affected by this means were not iden- 
tical to those altered by dietary restriction. 

Intermediary Metabolism 

The research activities of this section are con- 
cerned with the mechanisms of oxidative phos- 
phorylation associated with the respiratory chain 
and with the substrate level reaction associated 
with the a-ketoglutarate oxidation. 

The chemical reactions associated with the syn- 
thesis of ATP during oxidations in the mitochon- 
drial electron transport chain are not known. 
The work carried out clearly implicates a dithiol 



grouping in the process. The evidence rests on 
the observations that arsenite, under special con- 
ditions, uncouples oxidative phosphorylation in 
liver mitochondria, exposes an ATPase and in- 
hibits the exchange of P S2 -phosphate with ATP. 
The effects are quite analogous to those of 2,4- 
dinitrophenol. The involvement of dithiols in 
respiratory oxidative phosphorylation brings out 
the similarity of the process to the "substrate 
level" phosphorylation in the oxidation of a-keto- 
glutarate where the primary energy-trapping re- 
action involves the reduction of the disulfide 
group of lipoic acid and simultaneous formation 
of a thiol ester. A related mechanism, involving 
the oxidation of a charge-transfer complex be- 
tween a disulfide and a reduced electron carrier 
to the corresponding high-energy thiol derivative, 
has been proposed to explain the role of dithiols 
in the respiratory phosphorylation system. 

The mechanism proposed above is partly based 
on the results obtained in an investigation of the 
mechanism of the dihydrolipoyl dehydrogenase 
reaction. This enzyme has been identified as a 
navoprotein, and it carries out the terminal reduc- 
tion of DPN by dihydrolipoate in the sequence 
of reactions resulting in the oxidative decarboxy- 
lation of a-ketoglutarate. Based on the inhibition 
of the enzyme by arsenite and cadmium ions, the 
involvement of a second oxidation-reduction 
"prosthetic" group, namely, a disulfide-dithiol, 
has been identified. An interaction between the 
flavin-adeninedinucleotide and the dithiol results 
in the appearance of an absorption maximum at 
530 m^. in the reduced enzyme. A model for this 
interaction has been discovered in mixtures of 
reduced lipoate and flavinmononucleotide (FMN) . 
In the absence of any enzyme, the two compounds 
form a dissociable molecular complex with an 
absorption maximum at the same position. On 
standing, the complex breaks down to the lipoic 
disulfide and FMNH 2 . Neither the model com- 
plex nor the reduced enzyme show evidence for 
free radicals in the electron spin resonance spec- 
trum. The complex formed from reduced lipoate 
and FMN is analogous to the complex proposed 
in the scheme for respiratory oxidative phos- 
phorylation. Preliminary evidence suggests that 
the primary oxidation event in a-keto acid oxida- 
tion may involve an unknown grouping which 
comes into effect even before lipoate is reduced. 

The dynamic turnover of mitochondria in rat 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



livers has been studied. S 35 -methionine and C 14 - 
acetate were injected in the animals and the rate 
of loss of label followed in four mitochondrial 
components (soluble protein, insoluble protein, 
lipid and cytochrome c) . The results showed that 
all components had essentially the same half-life 
of 10.3 days which favors the conclusion that 
mitochondria are synthesized and broken down 
as units. The individual constituents do not ap- 
pear to be replaced after the synthesis of the 
particle. No difference in turnover rates has 
been detected in senescent (20-22 month) com- 
pared to adult (12 month) rats. 

Biophysics 

The research program of this section is directed 
toward an understanding of cellular mechanisms 
and their relationship to aging. At present at- 
tention is being directed toward cellular processes 
in cultures of the protozoan Euglena which can 
be artificially "aged" and to the relation between 
structure and function in muscle enzymes with 
special reference to the role of -SH groups. 

Experiments on the effect of steroid hormones 
on cultures of the protozoan Euglena have re- 
vealed items of importance. First, the increase 
in respiration caused by minute quantities of tes- 
tosterone is noticeable only in the actual presence 
of the hormone, and does not appear to persist 
after the cells are washed. Second, the effect 
of testosterone is not evident unless a source of 
nitrogen is in the medium. It thus finally appears 
possible to make an hypothesis on a biochemical 
or biophysical level — e.g., that testosterone con- 
trols membrane permeability. This hypothesis is 
further suggested by the finding that vitamin D, 
also a steroid, enhances the growth of these cells 
in a low Ca ++ medium, and work has now begun 
on the transport of Ca ++ into these cells in the 
presence and absence of added vitamin D. 

The effects of age and starvation on these pro- 
tozoan cultures have also been examined in a 
preliminary fashion. Viable cells have been 
maintained for many months in the absence of 
either a carbon or a nitrogen source. Preliminary 
indications were that drastic changes occurred in 
both the RNA and DNA content of the cells. 
Prolonged checking of the analytical procedures 
for DNA analysis has shown that the DNA 
analyses were not reliable and a series of minor 



modifications has been worked out which will now 
permit proper measurement of the DNA content 
at various times of "aging." The RNA content 
decreases from about 30 ^g/million cells to about 
5 (Ug/million cells or less. During the lag period 
that occurs when the cells are put into complete 
medium the KNA is resynthesized. The length 
of the lag has been found to increase with the 
time during which the culture was "aged" and 
some quantitative data on the relation between 
time of aging and length of lag have been ob- 
tained. When the cells are "aged" in a medium 
that contains no sulfur it is found that they can- 
not survive for more than about one month. Thus 
the mechanisms which permit the cell to survive 
in the face of nitrogen or carbon depletion do 
not function for the case of sulfur depletion. The 
existence of these adaptive regulatory mechanisms 
suggests that experiments on the content of cer- 
tain enzymes in these cells after various times of 
aging would be the most profitable direction for 
this work to proceed. 

The study of the role of -SH groups in muscle 
proteins has provided several new insights on the 
nature of the enzymatic process in myosin. Ear- 
lier in this project it had been shown that Cu + + , 
Cd ++ , and Zn++, like parachloromercuribenzoate 
(PCMB), would enhance the ATPase activity of 
myosin if these reagents were present in low con- 
centrations at high temperatures, e.g., 25° C) . At 
high concentrations, the ATPase activity was in- 
hibited, and at low temperatures inhibition oc- 
curred at any concentration of reagent. For 
ITPase activity only inhibition was observed. We 
have now studied the rates at which these reagents 
interact with myosin and the way in which the 
presence of the substrate — either ATP or ITP — 
affects this rate. From these studies it appears 
that there are at least two -SH groups on the 
active site. One -SH group is involved in the 
ability of myosin to differentiate between ATP 
and ITP, and may be involved in a small con- 
formation change at the site. Reaction of the 
second -SH group with any of these reagents 
leads only to inhibition. Studies on the tryptic 
digestion of myosin have shown that the active 
site is located on a trypsin resistant part of the 
myosin molecule and that the site does not inter- 
act appreciably with other parts of the myosin 
molecule. Several differences between Cu ++ and 
PCMB have also been found. In particular, the 



NATIONAL HEART INSTITUTE 



117 



use of Cu ++ at several pH values has revealed 
the presence of an ionizing group, with an appar- 
ent pK near 7.4, that influences the enzymatic 
activity. The results of this work on myosin sug- 
gested that the adenine part of the DPN coen- 
zyme molecule might interact with some dehydro- 
genase enzymes in a way similar to the interaction 
of the adenine part of ATP with myosin. Ex- 
periments with lactic dehydrogenase were begun 
with these hypotheses in mind. We have found 
a certain similarity between the effects of Cu + + , 
Ni ++ , Cd++, Co++, andZn++ on these two en- 
zymes, and, in addition, have succeeded in dem- 
onstrating that there is an -SH group on lactic 
dehydrogenase, the access to which is controlled 
not by the adenine but by the phosphate group 
on DPN which is closest to the nicotinamide 
portion of this coenzyme. Further work is in 
progress. 

Molecular Biology 

The principal concern of this section in the past 
has been the function of metal ions in biological 
processes. During the last year, however, the 
focus of attention has been an artificial reaction 
of metals with biological molecules in order to 
elucidate their structure. 

Structure of Nucleic Acids 

It is believed at the present time that all of 
the hereditary information that brings about the 
development of an organism as well as its degra- 
dative changes on aging is carried in molecules 
of deoxyribonucleic acid (DNA). The material 
consists of four basic building blocks, adenine, 
cytosine, guanine, and thymine, of which hun- 
dreds are joined together in a chain by means of 
ribose and phosphate units. The information 
code is believed to consist of the sequence in which 
these building blocks are arranged ; such sequence 
determination is therefore of utmost importance 
to an understanding of the hereditary code. At- 
tempts to determine this sequence have, up to this 
time, been confined to enzymatic degradations. 
Unfortunately, however, no enzymes are available 
for splitting specific bonds in nucleic acids. The 
present effort is to develop a chemical reaction 
capable of such specificity. 



Complexes of salicyl aldehyde with copper, 
nickel, and cobalt have been found to react with 
adenine to produce substances that are different 
and more stable than the substances produced 
from cytosine, guanine, and thymine. Using vari- 
ous combinations of metals, salicylaldehyde, and 
the four bases, their ribosides and their riboside 
phosphates, it is indeed possible to distinguish all 
of these substances qualitatively and some of them 
quantitatively by simple color tests. So far, this 
differentiation has been possible only for the 
molecules that have been disassembled from the 
chain. The metals have been found to react also 
with ribose, making the differentiation on the 
chain more difficult, but various techniques are 
being used to overcome this difficulty. 

Structure of Hemoproteins 

Meanwhile, studies on the relation of rotatory 
dispersion of the hemoproteins to their structure 
have continued. Hemoproteins are proteins con- 
taining an iron complex, heme; the hemoproteins 
catalase and hemoglobin contain four hemes per 
molecule, whereas peroxidase and myoglobin con- 
tain only one heme per molecule. The rotatory 
dispersion is a plot of optical activity vs. wave 
length; this plot exhibits a "Cotton effect," i.e., 
an S-shaped curve, in the visible when the heme 
group is asymmetrically placed, but the plot is 
essentially parabolic when the heme is not asym- 
metric. All four of the molecules, catalase, 
peroxidase, hemoglobin and myoglobin, exhibit 
the Cotton effect when the molecules are intact, 
and they lose the effect when the natural folding 
of the protein molecule is changed in some man- 
ner into a more irregular shape. In the case of 
catalase and hemoglobin, these changes were 
shown to be accompanied by splitting of the 
4-heme molecules into 1-heme quarters or 2-heme 
halves; here the phenomenon can be explained if 
each heme is symmetrically surrounded by protein 
and the presence of additional hemes in the mole- 
cule destroys this symmetry. However, in the 
case of peroxidase and myoglobin, the molecules 
cannot be broken down, and the rotatory disper- 
sion changes can be explained only if one accepts 
the following generalization: The Cotton effect 
associated with the heme absorption is a measure 
of the ordered arrangement of the protein and 



118 



ANNUAL EEVIEW OF INTRAMURAL RESEARCH 



disappears when the structure of the protein has 
been destroyed. 

This generalization has been applied in a study 
of the stability of the heme-protein site in cata- 
lase. The heme was removed and then added 
back to the protein. Four molecules of heme still 
reacted with each molecule of the catalase pro- 
tein, showing that the site on the protein, to which 
the heme had been attached, was not destroyed 
in this procedure. However, the newly reconsti- 
tuted hemoprotein showed no Cotton effect ; hence 
the structure of the protein as a whole has been 
destroyed. Therefore, the site for heme attach- 
ment remains intact even when the gross struc- 
ture of the protein is destroyed. 



Metal Ions in Enzymatic Reactions 

In the past we have studied the effect of metal 
ions on the substrates of enzymatic reactions. 
This year attention has been directed to the inter- 
action of metals with proteins, specifically bovine 
plasma albumin (BPA). The most striking ob- 
servation has been that the BPA molecule dimer- 
izes in the presence of copper, but is not affected 
in this way by any other metal. Also, copper 
and cobalt shift the equilibrium between two 
forms of BPA, although manganese has no effect. 
A possible explanation of the specificity of man- 
ganese in many enzymatic reactions is that man- 
ganese, unlike other metals, will not change the 
structure of the protein. 



NATIONAL INSTITUTE OF ALLERGY 
AND INFECTIOUS DISEASES 



INTRODUCTION 

The year 1960 has seen both a widening of the 
scope of research activities and an intensification 
of the pursuit of promising lines of investigation 
in the National Institute of Allergy and Infec- 
tious Diseases. The coming year will see even 
more effective utilization of scientific resources, 
as new space becomes available and programs 
initiated this year begin to achieve their objec- 
tives. 

Clinical investigations of the Institute have 
advanced in several ways. Of great significance 
for present and future programs has been the 
increased utilization of prisoner volunteers for 
clinical studies, through cooperation with the 
Federal Bureau of Prisons. Volunteers have been 
hospitalized in the Clinical Center, exposed to 
specific respiratory viruses, observed for clinical 
manifestations, and examined by precise labora- 
tory techniques for evidence of infection. In a 
short time, these human volunteer studies have 
made available a vastly greater amount of de- 
tailed information concerning the respiratory syn- 
cytial virus and the Eaton agent than would have 
been possible by the usual clinical and epidemio- 
logical observations in the general population. 

In connection with investigations of simian 
malaria, prisoner inmates have been inoculated 
with various strains of the parasite in the Atlanta 
Penitentiary. Some of them were transferred to 
the Clinical Center for precise clinical observa- 
tions. This study also has permitted the extension 
of clinical and laboratory observations not pos- 
sible in any other way. Other clinical studies 
underway for some years such as treatment of 
fungus infections, bacterial complications of cys- 
tic fibrosis, and drug resistance in staphylococcal 
infection have continued to add to specific knowl- 
edge of these diseases. 

During the year an observation of great poten- 
tial importance to public health was made by 



Institute malaria investigators and quickly sub- 
stantiated by two other laboratories. Two scien- 
tists working with a strain of monkey malaria 
were accidentally infected, probably by a mos- 
quito bite. This was the first clear-cut evidence 
that certain strains at least, of simian malaria, 
are pathogenic for man. An intensive investiga- 
tion using all available resources quickly showed 
that man could be repeatedly infected by this 
strain through mosquito bite, that it could be 
transferred from man to man by blood, and that 
monkeys could be infected by mosquitoes fed on 
human cases. These observations formed the 
basis of a greatly expanded program on the 
pathology and biology of malaria, including the 
establishment of a field party in Malaya, the 
origin of the monkey strain infectious for man. 

The Middle America Research Unit in Panama, 
established three years ago under the sponsorship 
of this Institute and the Walter Reed Army In- 
stitute of Research, has intensified its studies of 
the arthropod-borne viruses in the tropics. Labo- 
ratory procedures have been developed and diag- 
nostic reagents prepared for working with a large 
proportion of the more than 125 arthropod-borne 
viruses. The Middle America Research Unit also 
has extended its studies to include special epi- 
demiologic observations of poliomyelitis in Pan- 
ama, and investigations of the role of mites in 
Central American virus infections. In conjunc- 
tion with Gorgas Memorial Laboratory workers, 
Institute scientists recovered two strains of vesic- 
ular stomatitis virus from Phlebotomua flies. 
This is the first time this group of insects has 
been incriminated as a possible vector of this 
important virus. 

Increasing interest in immunologic phenomena 
manifests itself in nearly all aspects of Institute 
research activities. The development of new im- 
munologic techniques, such as immunoelectropho- 
resis and immunofluorescence, has stimulated new 



119 



120 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



approaches to new and old questions and attracted 
investigators into the field. The result has been 
a noticeable resurgence of clinical and scientific 
interest in allergic diseases. The Institute initi- 
ated a new program in clinical immunology dur- 
ing the year which will investigate such auto- 
immune diseases as lupus erythematosus, and 
thyroiditis, as well as certain clinical aspects of 
hypersensitivity and mechanisms of resistance. 

The very important studies on respiratory in- 
fections which have been underway for some 
years continue. It is now clear that the Eaton 
agent is, as long suspected, an important cause 
of primary atypical pneumonia. Development of 
more precise laboratory techniques for isolation 
of viruses from animals, demonstrated that many 
laboratory mouse stocks are grossly contaminated 
with normally occurring latent viruses. At least 
five different viruses are now known to infect 
apparently normal mice. This has complicated 
studies on the relation of viruses to cancer, and 
confused much of the work in this field conducted 
over the last few years. It seems unavoidable 
that the development of pathogen-free animal 
stocks, particularly mice, will be essential for 
future investigations in cancer and in other dis- 
eases as well. The development of some means 
to eliminate or control these contaminating viruses 
from experimental animals poses a major and 
immediate problem to this Institute and to in- 
vestigators elsewhere. 

The year has witnessed also the planned de- 
velopment of research experience for junior inves- 
tigators and of opportunities for permanent staff 
members to work in research centers outside the 
National Institutes of Health. The key to pro- 
ductive and significant research is, as has been 
pointed out repeatedly, the selection of imagina- 
tive, energetic and well trained investigators and 
the development of a stimulating environment in 
which these investigators have the freedom and 
resources to pursue their scientific ideas. One 
approach to this goal is to recruit competent in- 
terested young men soon after completion of their 
doctoral training. During the last year three 
such young men joined this Institute as a part 
of the NIH Kesearch Associate Program and 
seven as part of the Clinical Associate Program. 
Six others were recruited to specialized research 
activities making a total of 16 new scientific 
staff members at the junior level. From these no 



doubt will come a number of our future perma- 
nent staff and all will become indoctrinated with 
research experience of great value to their even- 
tual scientific development. Thus, the intramural 
activities perform an important role for training 
in research methods and goals to the future bene- 
fit of scientific endeavor, wherever these men may 
work — in universities, hospitals, or in the prac- 
tice of medicine. 

The Institute has pursued vigorously a policy 
of encouraging work assignments of its perma- 
nent staff in other well known research centers. 
One scientist is working at the Karolinska Insti- 
tute^ Stockholm, Sweden ; two are at the Pasteur 
Institute, Paris, France; one at the Max-Planck 
Institute for Virus-forshung, Tubingen, Ger- 
many; one in collaboration with the University 
of California and Institut de Recherches Medi- 
cales de la Polynesie Francaise, Papeete, Tahiti; 
one at the Wallaceville Animal Research Station, 
Wellington, New Zealand ; and one with the Virus 
Laboratory, California State Health Department. 
Others have spent shorter periods of time in 
Malaya, Africa, and Brazil, pursuing specific re- 
search problems. A number of the staff have 
served on WHO Expert Committees and on spe- 
cial assignments, particularly in the areas of 
tropical medicine and parasitology. In this field, 
this Institute has probably the largest group of 
investigators of any other center in this country. 

Research in tropical diseases will be expanded 
still further under the provisions of Public Law 
480 which permits the use of foreign currencies 
to support specific intramural research projects 
in certain countries. Projects have been formu- 
lated and implementation of them during the 
coming year will permit the selected expansion 
of investigations important on a worldwide scale. 
These include problems in such diseases as ma- 
laria, schistosomiasis, fungus infections, arthro- 
pod-borne viruses, and filariasis. These interna- 
tional activities emphasize the expanding scope 
of our research responsibilities. 

The Board of Scientific Counselors met twice 
during the year. The first meeting considered 
Institute investigations underway on respiratory 
diseases of viral etiology. The second meeting 
was held at the South Carolina State Hospital, 
Columbia, South Carolina where the Epidemi- 
ology Section of the Laboratory of Parasite 
Chemotherapy is located. Institute staff members 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



121 



reviewed studies on simian malaria, chemother- 
apy of malaria, and intestinal parasite infections. 
This report arranged by laboratory activities 
will summarize the major scientific accomplish- 
ments and advances achieved during the year in 
the direct intramural program. 



Approximately 24 other volunteers have par- 
ticipated in studies with para-influenza 4 virus 
or Eaton (primary atypical pneumonia) agent. 
Future studies are planned with human influenza 
virus and with the recently defined group of 
REO viruses. 



LABORATORY OF CLINICAL 
INVESTIGATION 

Clinical research activity has expanded during 
the past year largely as a response to enlarging 
the professional staff and cooperation with other 
Institute research units. A further period of 
growth will be needed to staff the clinical service 
in a manner consistent with optimum research 
productivity. 

Infection of Volunteers with Respiratory Viruses 

An extensive clinical study of acute viral res- 
piratory disease was begun this year in associa- 
tion with staff members of the Laboratory of 
Infectious Diseases. The initiation of this project 
required much work but with the unstinting as- 
sistance Of the Clinical Center, very satisfactory 
arrangements have been made to transfer to the 
Clinical Center, Federal prisoner-volunteers for 
study. This has been done with the permission 
and considerable assistance of Mr. James Bennett, 
Director, and Dr. Harold Janney, Medical Direc- 
tor of the Bureau of Prisons, Department of 
Justice. A team of custodial officers has also 
been assigned to the Clinical Center by the Bu- 
reau of Prisons to oversee the volunteers. The 
volunteers have uniformly cooperated with the 
program despite some extended periods of room 
isolation, frequent blood-letting, and other incon- 
veniences. Many administrative arrangements 
have been developed so that the program has 
worked increasingly smoothly. 

The results so far justify the investment in 
money and effort. It has been possible to produce 
in human volunteers a rather uniform "cold" with 
the respiratory syncytial virus. It may occur 
without respect to preinfection immunity, but the 
subsequent rise in complement-fixing antibody 
appears to correlate with severity of illness. 
Forty-six men have so far participated in this 
study. 



New Antifungal Drug 

Beginning about four years ago, the Mycology 
Section of the Laboratory of Infectious Diseases 
began studies on an antifungal drug produced by 
Hoffman La Roche, designated as RO-2. This 
agent, an antimicrobial, was found to be the most 
active material ever tested in vitro and in animals 
against several of the pathogenic fungi, notably 
histoplasmosis and blastomycosis. 

In the intervening years, 30 patients have been 
treated in the Clinical Center. Extremely favor- 
able results have been observed in several patients 
severely ill with these diseases. From the stand- 
point of therapeutic activity, it appears that this 
drug is the best available for blastomycosis and 
histoplasmosis. 

During the studies it was noted that the agent 
produced unusual hepatic changes. It was found 
that the dye, bromsulphalein, normally rapidly 
transferred from the blood to the bowel by the 
liver, was retained in high concentration in the 
blood in patients treated with RO-2. This effect 
appeared in a day or two after start of treatment, 
before tissue changes would likely occur, suggest- 
ing competition of the new drug for the liver 
excretory mechanism for bromsulphalein. After 
treatment was stopped, dye excretion promptly 
returned to normal or nearly normal. Liver 
biopsy has revealed changes indicative of minor 
hepatic damage in some cases. Because of the 
great importance of having a drug in addition 
to the relatively toxic agent, amphotericin B, for 
the treatment of fungal diseases, this new agent 
continues under investigation. 



Simian Malaria 

Following the demonstration of the infectivity 
of Plasmodium cynomolgi bastianellii for man, 
several inmates at the Federal prison in Atlanta 
volunteered for exposure to this agent. The need 
for careful clinical characterization of this dis- 
ease in man led to the transfer of two infected 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



volunteers to the Clinical Center. Both men de- 
veloped acute malaria which was carefully studied 
throughout its course. Significant alterations in 
urinary steroid excretion were detected, an un- 
usual elevation of serum cholesterol occurred in 
one, and liver lesions not previously described, 
resembling but not identical with exoerythrocytic 
phase parasites, were demonstrated in both pa- 
tients. 



Penicillins and Penicillinase 

Previous work here had defined nutritional re- 
quirements for penicillinase production by staph- 
ylococci and many parameters of its interaction 
with benzyl penicillin (penicillin G) . During the 
year English investigators, working with the 
stripped molecule of penicillin, 6-amino-penicil- 
lanic acid, produced a new compound largely 
resistant to destruction by penicillinase. Work- 
ing with this and several other penicillin deriva- 
tives, it was found that resistance to penicillinase 
is greatly influenced by steric positions of ethoxy 
groups on the side chain and that failure to be 
destroyed by penicillinase is associated with 
greater penicillinase inducing-capacity. Perhaps 
most significantly, this work has added to the 
now substantial indirect evidence that the major 
reason for present resistance of staphylococci to 
penicillin is the capacity of these micro-organisms 
to produce penicillinase upon contact with even 
low doses of penicillin. 

Clinical studies with the penicillinase-resistant 
penicillin, dimethoxyphenyl-penicillin, have re- 
vealed that it possesses resistance to penicillinase 
in vivo, and that it is a powerful and effective 
anti-staphylococcal drug. After treatment of 
some patients for periods of three to five months, 
no penicillin-resistant staphylococci have been 
isolated. It has long been considered that chronic 
staphylococcal infection resembles tuberculosis 
and for the first time it appears that an agent 
is available which can be employed for extended 
periods of treatment without loss of effect, such 
as isonicotinic acid hydrazide in tuberculosis. If 
long-term therapy can thus be regularly given 
an enormous benefit will accrue to thousands ill 
with chronic staphylococcal disease. 



Ascites in Mice By Injection of Adjuvants 

In the past year a staff member has continued 
to study ascites induced in mice by the injection 
of adjuvant mixtures. This procedure has pro- 
vided a much needed laboratory method for pro- 
ducing antibodies of a wide variety and a means 
of evaluating antigens. Eecently, interest has 
been focused on the pathology and abnormal 
physiology of the lesion. It has been found that 
strain differences are associated with differences 
in susceptibility to ascites. Since ascites was 
found associated with a local plasma cell reaction, 
which in some strains of mice went on to plasma 
cell tumors, many implications toward problems 
in neoplasia have also been raised. These studies 
have become the basis for biochemical and patho- 
logic studies with other Institutes. 

Hypogammaglobulinemia 

Staff members have shown that patients with 
hypogammaglobulinemia possess low, but defi- 
nitely measurable, levels of antibody to the enteric 
viruses. The implication of this finding, in view 
of the normal resistance of these patients to viral 
infections, is that extremely low levels of specific 
antibody may provide adequate resistance against 
viral diseases. Subsequent, unpublished studies 
have indicated that these patients will develop 
circulating antibodies to Salk polio vaccine. 

Bentonite Flocculation Test 

The modification of the bentonite flocculation 
test for the detection of gamma globulin promises 
to provide the practicing physician with an ac- 
curate, convenient laboratory aid in the diagnosis 
of hypogammaglobulinemia. In contrast to the 
electrophoretic method, the results with the ben- 
tonite test can be known to the physician within 
a few minutes of arrival of the specimen to the 
laboratory. Other modifications of this technique 
will also give the levels of albumin and other 
protein constituents of blood and other fluids 
without resort to the more cumbersome method 
of electrophoresis. 

The DNA-bentonite test for systemic lupus 
erythematosus has also been developed in the 
past year. This test measures the antibody in 
lupus serum which is directed against nuclear 
material. The specificity of this test is greater 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



123 



than any previously described test for lupus. This 
test, however, is positive primarily in those pa- 
tients with active disease and only rarely is posi- 
tive when the disease is in remission. A modifi- 
cation of this test, the nucleoprotein-bentonite 
test, has retained all the attributes of the DNA 
test in regard to specificity, while achieving a 
much higher level of sensitivity for cases in re- 
mission. These promise to become important 
standard tests in the diagnosis of systemic lupus 
erythematosus. 

Cystic Fibrosis of the Pancreas 

Despite the commonly accepted point of view 
that antibiotics are helpful in this disease, obser- 
vations of nasopharyngeal cultures failed to re- 
veal any appreciable effect of antimicrobial treat- 
ment on. Staphylococcus aureus. This does not 
negate a possible clinical benefit but does appear 
to minimize its value. It was coincidentally ob- 
served that Escherichia coli was not present in 
the nasopharyngeal flora of any child over the 
age of eight. 



ROCKY MOUNTAIN LABORATORY 

At the Rocky Mountain Laboratory research 
has continued directed toward both basic labora- 
tory investigations and field studies of insect- and 
animal-borne diseases. In the first category are 
those projects concerned with the chemistry and 
surface properties of viruses, the highly in- 
triguing relations of hypersensitivity and hu- 
moral immunity, and the basic relation of struc- 
tural elements of microorganisms to the activity 
of the agents, both in vivo and in vitro. Field 
work is the foundation of projects related to 
studies of Q fever, tularemia, Colorado tick fever, 
and the ar-bo viruses. In addition, the combined 
efforts of the staff are directed to many other 
areas of research. Such projects include tuber- 
culosis, influenza, poliomyelitis, and cryptococ- 
cosis. 

Hypersensitivity 

Studies of hypersensitivity at the Rocky Moun- 
tain Laboratory have been directed primarily 
toward clarification of the relations existing be- 



tween delayed hypersensitivity and circulating 
antibodies and determination of the factors re- 
sponsible for induction of contact hypersensitiv- 
ity. It has been demonstrated previously that 
delayed hypersensitivity precedes circulating anti- 
bodies. This delayed hypersensitivity is directed 
toward protein. When circulating antibody ap- 
pears, as occurs with relatively large amounts of 
conjugated protein, or when booster doses are 
administered, the specificity of the antibody re- 
sponse becomes oriented toward small configura- 
tions on the antigen molecule. Studies of im- 
munity in neonatal animals have revealed that 
these animals, when injected with an antigen 12 
hours after birth, develop circulating antibodies 
but fail to develop delayed hypersensitivity. Ad- 
ditional experiments suggest that this inability of 
neonatal animals to express such reactions is due 
to a deficiency in a skin reactive factor rather 
than an inability to respond to a primary injec- 
tion of antigen. 

Poliovirus 

Continued studies of poliovirus have yielded 
considerable fundamental data. In cooperation 
with the group at the University of Minnesota, 
it was shown that agents such as octyl alcohol- 
chloroform and neutral hydroxylamine do not 
materially change the physical properties of puri- 
fied infectious RNA of poliovirus but destroy 
over 99 percent of the infectivity of this material. 
The destruction of infectivity by uncoupling of 
a single link in a large particle (probably an acyl 
link of an amino acid with a phosphate group 
at the end of an RNA chain) suggests a possible 
approach to virus chemotherapy. Other studies 
have revealed that only 0.1 percent of RNA in- 
fectivity could be accounted for by residual pro- 
tein, thus strengthening the concept that RNA 
does indeed constitute the infectious portion of 
the poliovirus moiety. By the use of chromato- 
graphic methods, it was also demonstrated that 
only certain of the avirulent strains of poliovirus 
can be differentiated from the virulent strains 
from which they are derived. This is in direct 
contrast to work reported by others. In studies 
of the infectivity of RNA it was found that the 
bulk of virus particles react with susceptible cells 
and that the relatively poor correlation between 
virus particles and PFU is due to the poor effi- 



124 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ciency of RNA at entering sites where it can in- 
fluence virus production. A precipitation test 
for detection of antibodies against poliovirus has 
been developed which is more sensitive than the 
neutralization test presently employed. The an- 
tigen used is radioactive virus. 

Endotoxins in Bacterial Fractions 

Research on endotoxins derived from Gram- 
negative organisms has continued. As is so fre- 
quently the case, a fresh outlook on an old prob- 
lem yields results of great value. The ideas held 
by Dr. Westphal, which attributed the activity 
of endotoxins to the presence of a firmly bound 
lipid ("lipid A"), were apparently generally ac- 
cepted until presentation of the work done at 
RML. The finding that lipid A was not active 
and that deproteinized and "delipified" endotoxin 
was active stirred considerable controversy. In 
fact, the controversy was so intense that efforts 
to develop a vaccine against Salmonella infec- 
tions have been diverted to settling this issue. 
Recent studies of the kinetics of inactivation of 
toxin by hydrolysis with hot acid have given data 
which should end this discussion. The old con- 
cept of "purified endotoxins" must be abandoned 
since there have been no previous toxins as good 
as those obtained at RML, and these are to be 
purified still further. 

The purification of Vi antigen by curtain elec- 
trophoresis is a major advance in the study of 
this most important antigen. The demonstration 
that certain labile acetyl groups are responsible 
for the activity of Vi antigen resulted in produc- 
tion of material which was ten times more active 
than purified preparations prepared by mild acid 
hydrolysis. Emphasis should be placed upon the 
new chemical, physical, and biologic methods that 
have been devised to solve these problems. 



Tuberculosis 

The problem of immunity in tuberculosis has 
been studied intensively. Significant findings in- 
clude the fact that mice may be satisfactorily 
immunized to subsequent pulmonary infection 
with virulent organisms by administration of 
small doses of avirulent organisms by the aerosol 



method. The demonstration that resistance is 
not due to interference strengthens the case for 
the value of immunization with living attenuated 
organisms for the prevention of tuberculosis. 
Since it has been shown that the delayed reac- 
tions elicited by protoplasm of various acid-fast 
organisms are specific in nature, it seems prac- 
ticable to apply these findings to certain diagnos- 
tic problems in man. Use of fractions of tubercle 
bacilli in producing isoallergic encephalitis in 
guinea pigs show that the adjuvant effect lies in 
the cell walls and in a water-soluble protein pre- 
pared from the walls. This latter finding is im- 
portant since it will allow the study of the adju- 
vant phenomenon from a molecular level. 



Q Fever 

The number of dairy cattle infected with 
Coxiella bumetti is large and is increasing, yet 
it is extremely difficult to detect cases of Q fever 
in man. In Idaho and Montana we have shown 
that a greater number of individuals residing on 
infected premises have antibodies against this 
organism than do those living on noninfected 
premises, yet no difference can be detected in the 
number of individuals who have symptoms com- 
patible with clinical disease. The fact that or- 
ganisms isolated from cattle have been uniformly 
of low virulence for experimental animals may 
account for the inability to find clinical cases of 
disease in those exposed only to cattle. 

By the use of skin tests to eliminate allergic 
individuals from the study group, 190 inmates 
of the Montana State Prison were safely im- 
munized without producing such reactions as 
have been previously reported. It is evident from 
these results, as well as from those previously 
obtained in laboratory personnel, that human be- 
ings can be safely vaccinated against Q fever if 
the precaution is taken to eliminate reactors by 
previous administration of specific skin-test an- 
tigen. 

Methods developed for growing rickettsiae on 
modified Zinsser tissue cultures yielded relatively 
large volumes of organisms. These studies led to 
others involving purification of G. bumetti by 
sucrose gradients and by continuous-flow centrifu- 
gation in molar salt solution. These methods 
likewise made it possible to obtain certain chemi- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



125 



cal and physical fractions of these organisms. It 
was found that dimethyl sulfoxide could extract 
from Phase II C. burnetii a material which acted 
only as a hapten, but from Phase I organisms 
the extract obtained acted as a complete antigen. 
Lauryl sulfate also extracts complete antigen 
from Phase I organisms. Physically, the cell 
walls of these organisms can be separated from 
the protoplasm, and it has been noted that the 
cell walls are about 25 times more active in pro- 
ducing immunity than is protoplasm. 

These studies on Q fever are of considerable 
significance. The laboratory and related studies 
have yielded information of both scientific and 
applied interest. It is apparent now that we can 
safely use our present vaccines for immunization 
of man and that it is feasible to produce large 
numbers of organisms which can be purified and 
used as vaccine or manipuated to give physical 
or chemical fractions which may be less toxic. 
The failure to find clinical cases of Q fever in 
man in the face of a rising incidence of infection 
in dairy cattle is highly interesting even if dis- 
appointing. 

Other Rickettsioses 

Studies of rickettsiae other than C. burnetii 
have been continued. By combining the methods 
presently used for fluorescent microscopy, a tech- 
nique for sectioning arthropods has been devel- 
oped which should be of interest to entomologists 
working in the field of embryology and anatomy 
and to medical entomologists, since thin sections 
in which the organs are not displaced can be 
obtained routinely. By applying the technique 
to the study of ticks infected with R. rickettsii 
it was found that the infection rate in local ticks 
varies from 15 to 28 percent. Not all of the ticks 
found infected by this method are infective for 
laboratory animals. The value of this type of 
study has yet to be fully appreciated. 

The use of specific toxins has resulted in clari- 
fication of many of the problems related to the 
taxonomy of rickettsiae and has proved to be 
useful in ecological and epidemiological studies 
of this complex group of diseases. In further 
studies, potent immunogenic extracts have been 
obtained from certain of the rickettsiae. Their 



value as diagnostic and prophylactic agents is 
presently under consideration. 

Bacterial Vaccines 

Studies have been continued on vaccines for 
certain bacterial diseases. It has been found that 
while live Russian tularemia vaccine is capable 
of protecting mice more effectively than does 
ether-extracted vaccine derived from cell walls 
of Pasteurella tularensis, the protection produced 
by live organisms was not effective for long 
periods of time. Continued studies have empha- 
sized the value of cell walls in producing im- 
munity to infections with Brucella abortus in 
laboratory animals. It has also been found that 
live cells suspended in phosphate buffer and 
shaken with an excess of ether are killed but not 
disrupted. These cells constitute an excellent 
protective antigen which is less toxic (LD 50 7.5 
mg.) than aqueous ether extracts obtained by 
conventional methods (LD 60 0.9 to 2.0 mg.). 

Arbor Viruses 

Studies of arbor viruses have yielded results of 
interest and suggest that emphasis on field studies 
would greatly increase the production of useful 
data. The California strain, described by Reeves 
and Hammon, has been isolated from a snowshoe 
hare in Montana, and serologic studies of hares 
obtained from Michigan indicate that the major- 
ity possess antibodies against this virus. In Cali- 
fornia it has been demonstrated that, although 
most infections in man with this agent are of 
the inapparent type, some infections result in 
serious disease. A virus closely related to 
Powassan virus was recovered from ticks from 
Colorado and is of importance since viruses of 
this group produce serious illness in man. Studies 
to date indicate that ticks probably are not the 
natural vector, but the relation to Powassan virus 
suggests that mosquitoes would most likely be the 
vector in nature. In studies of the complex rela- 
tion of WEE virus with snakes and mosquitoes 
it has been possible to demonstrate that the virus 
can be readily overwintered in garter snakes and 
that mosquitoes can be infected by feeding on 
such snakes. While we have not been successful 
in isolating virus from snakes collected in the 
field, the laboratory data suggest that these or 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



similar animals could constitute a host suitable 
for overwintering of WEE virus. In Idaho and 
Oregon, WEE virus was isolated with consider- 
able frequency during the summer season, while 
in North Dakota the virus did not appear to be 
active. Isolations of a considerable number of 
strains of trivittatus and inornata viruses were 
made. 

Considerable research was performed to deter- 
mine the level of viremia attained in wild and 
domestic birds infected with arbor viruses. After 
infection with WEE or St. Louis viruses, turkeys, 
ducks, chickens, and pheasants display levels of 
viremia which should cause infection in mos- 
quitoes feeding on them. It is of interest, how- 
ever, that in spite of considerable effort we have 
been unable to isolate arbor viruses from the 
bloods of vertebrates. Negative results were ob- 
tained in examination of 1,074 specimens collected 
in Montana, North Dakota, Oregon, and Minne- 
sota during the spring of 1960. These studies 
fail to add weight to the contention that latent 
infections of birds are a factor in overwintering 
or of introduction of virus into endemic areas. 

Colorado Tick Fever 

Colorado tick fever continues to be a problem 
in the western United States. Without stimula- 
tion of physicians a large number of specimens 
for examination were received this year and virus 
was isolated from 49 of them. Our interest in 
the spectrum of symptoms has continued and we 
still see severe cases of illness due either to en- 
cephaliticis or bleeding tendencies. It was found 
that the complement-fixation reaction developed 
at the Eocky Mountain Laboratory is the simplest 
method for diagnosis of Colorado tick fever. 
Vaccine has been prepared and has been shown 
to be efficacious in mice. This type of vaccine 
has been used repeatedly in man without ill ef- 
fects, indicating that a vaccine prepared from 
suckling mouse brain is harmless to man when 
repeated doses are given. 

Ticks collected in Estes Park, Colo., were ex- 
amined for the presence of Colorado tick fever 
virus. The incidence of infection was found to 
vary from 5 to 21 percent. This high incidence 
of infection in ticks accounts for the large num- 
ber of cases of CTF reported in Colorado annu- 
ally. 



LABORATORY OF TROPICAL VIROLOGY 

The activities of this laboratory are conducted 
at the Middle America Research Unit in the 
Panama Canal Zone and at Bethesda. The Mid- 
dle America Research Unit is a joint research 
effort of the National Institute of Allergy and 
Infectious Diseases which has cognizance for 
studies on virus diseases and the Walter Reed 
Army Institute of Research which has cognizance 
for studies on fungus diseases. 



Virus Isolates 

During the first 12 months of a 3-year project 
on the ecology of arthropod-borne viruses in the 
tropical rain forest, conducted by the Gorgas 
Memorial Laboratory with the collaboration of 
MARU, major emphasis has been on virus isola- 
tion in suckling mice and hamster kidney cell 
cultures. Fourteen virus strains were isolated at 
MARU from 412 pools and 63,000 specimens pro- 
vided by GML. Virus isolation rates were for 
Phlebotomus, 1 :700 and for mosquitoes, 1 :7000, 
although the rates varied greatly with species. 
Of the five Phlebotomus isolates, two of broad 
host range (including cell culture) and short 
incubation period are serologically identical. 
These viruses have now been identified as the 
Indiana type of vesicular stomatitis virus. The 
other three Phlebotomus and nine mosquito vi- 
ruses are being related to each other, to known 
virus groups and to human and/or animal infec- 
tion and disease. 



Eastern Equine Encephalomyelitis 

The prevalence of EEE antibodies in horses 
and man in two areas of suggested EEE virus 
endemicity has been determined, allowing an 
evaluation of the relative usefulness of several 
serological methods applicable to studies of this 
type. It was found that the incidence of EEE 
antibodies in 460 humans tested increased with 
advancing age (0.8 percent) under 10 years with 
progressive increase to 9 percent in the 41-50 
year group) . Complement fixation results on the 
same sera indicated the probable presence of other 
group A viruses. 

Lizards of species common to this part of Pan- 
ama were examined as a possible virus reservoir. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



127 



Specific EEE virus hemagglutination-inhibitors 
were found in some of their sera. The occurrence 
of viremia and HI antibody response following 
virus inoculation were experimentally confirmed 
by inoculation of lizards. 

Encephalomyocarditis 

Previously this laboratory described an out- 
break of a fatal disease of swine caused by the 
EMC virus. The outstanding lesion in pigs dying 
during the outbreak was acute myocarditis. Since 
epidemiological observations suggested that natu- 
ral infection resulted from ingestion of contami- 
nated food, experiments were undertaken to re- 
produce the disease by feeding virus to young 
pigs. Viremia and virus excretion from the gas- 
trointestinal tract were found to occur following 
the administration of brain from EMC inoculated 
mice. Infected pigs developed high titers of HI 
and neutralizing antibody during convalescence 
and had myocardial fibrosis at autopsy. Other 
studies included demonstration of EMC anti- 
bodies in a small number of city rats and rats 
caught on the affected farm, although wild ro- 
dents were found to be negative. Human sera 
were examined with interesting differences in the 
results depending on the donors' age : while a 
substantial proportion of the Panamanian popu- 
lation has been infected with EMC virus, the 
antibodies were found to be more common in 
persons of younger age. 

Enterovirus Flora in Central America 

For a period of 12 months the enterovirus flora 
of infants at an outpatient clinic in Panama City 
was systematically explored, establishing a base 
line of enterovirus fluctuation. The majority of 
viruses isolated belonged to the ECHO group, 
although in late 1959 and early 1960 poliovirus 
type 2 had become very prevalent. This was re- 
flected in an uncommon occurrence of a small 
outbreak of paralytic disease due to type 2 polio- 
virus. 

Other enterovirus studies have included 1) sur- 
veillance for the presence of type 1 poliovirus in 
Panama in late 1960 as a check on dissemination 
and threatened spread of this commonly epidemic 
type, 2) studies on a major epidemic of ECHO-9 
virus which swept through the Republic of Pan- 



ama and the Canal Zone and 3) initiation of a 
collaborative project on possible relation of en- 
terovirus flora of Guatemalan children to their 
dietary status. 

Mycotic Diseases in Panama 

The research program on mycotic diseases has 
markedly increased local awareness of histoplas- 
mosis in all of its clinical forms, as evidenced by 
recognition of three disseminated cases, two fatal 
and one successfully treated, within a period of 

18 months. Until then only one fatal case had 
been described since Darling's original cases in 
1906. Ecological and epidemiological studies led 
to isolation of H. capsulatum from eight addi- 
tional soil samples, bringing up to 16 the total 
number of recent isolations from Panamanian 
soil. The fungus has been repeatedly recovered 
from the organs of trapped ground mammals, 
confirming its wide dissemination in nature. 

Histoplasmin skin test continues to be a major 
tool for the study of epidemiology of histoplas- 
mosis. Data on 9,200 children between six and 

19 years of age have been obtained indicating, 
as expected, that the percentage of reactors in- 
creases progressively with age. The rate of histo- 
plasmin sensitivity varies from 13 to 58 percent 
among 6-year-olds and from 68 to 92 percent 
among 19-year-olds, depending on location of 
their residence. A survey of 631 preschool chil- 
dren (6 months to 6 years) in the Canal Zone 
demonstrated an increase in hypersensitivity be- 
ginning with 3 years of age. A continuing simi- 
lar study of Panamanian children in a city hos- 
pital is now in progress with information on over 
800 already available. 

Projects on other mycotic diseases have in- 
cluded diagnostic study and therapy of monilia- 
sis, found to be a major superficial mycosis among 
both indigenous and transient population in the 
tropics. 

Arbor Virus 

At Bethesda new projects involved an inter- 
esting application of the technique of antiserum 
pool combinations to typing of arthropod-borne 
viruses, a wealth of data evaluating experimen- 
tally produced EEE virus infection in horses and 
a promising attempt to develop an inactivated 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



EEE virus vaccine for human use. The infected 
horses yielded specific antiserum which is being 
processed for prophylactic use in cases of human 
exposure under laboratory or natural conditions. 
Accidental laboratory infection of a staff mem- 
ber with an arthropod-borne group C(Apeu) 
virus led to the first clinical-virological study of 
a syndrome produced by this important and 
common group of viruses of the western hemi- 
sphere. 



LABORATORY OF BACTERIAL DISEASES 

The research program of the Laboratory of 
Bacterial Diseases has continued in the same 
general areas as last year. 

Intracellular Parasitism 

These studies deal with possible changes in 
characteristics of infected and immune cells as 
the result of parasitism, and the effect of intra- 
cellular growth on the parasite. One such notable 
change of course is the production of specific 
antibodies by certain cells of immune animals. 
Effort has been directed toward the study of an- 
tibody production by cells in vitro and the macro- 
phage was selected as a multipotential cell for 
such study. Macrophages obtained from the 
peritoneal cavity of guinea pigs immunized with 
egg albumin have been found to release antibody 
in vitro for a period of several days. This pro- 
vides a system for further study of the nutri- 
tional or other requirements for continued anti- 
body production in vitro, or even in serial 
cultures. Cells derived from macrophages have 
been carried in serial tissue culture for several 
months, retaining their phagocytic ability. 

Brucellosis 

Studies on brucellosis are conducted at a re- 
duced tempo. There is continuing need to col- 
laborate with other brucellosis research centers 
throughout the world to try and settle problems 
of classification and epidemiology of the Bru- 
cella. Currently we are doing some laboratory 
testing of brucellosis vaccine for human use pre- 
pared in Russia. There is present interest in this 
vaccine by the World Health Organization for 



its possible use in occupational and otherwise 
continually exposed groups. 

Studies on the Staphylococcus are directed 
toward determining the factors responsible for 
pathogenicity, and toward development and 
standardization of tests for measuring relative 
pathogenicity of strains. 



LABORATORY OF CELL BIOLOGY 

The activities of the Laboratory of Cell Biol- 
ogy during the calendar year 1960 have been 
along three major lines: (A) The continued ex- 
ploration of the metabolism of normal cultured 
cells, and an approach to the problem of meta- 
bolic controls; (B) the mechanism of viral syn- 
thesis; and (C) the study of cell cultures deriv- 
ing from patients with hereditary metabolic 
disease. 



Metabolism of Normal Cultured Cells 

A number of significant observations have been 
made with respect to the amino acid metabolism 
of cell cultures. There has been no further elu- 
cidation of the pathway of serine synthesis; but 
the mechanism of crystine synthesis has been 
clarified, in that all the cell lines so far studied 
have been shown to use the classical pathway 
involving the demethylation of methionine to 
homocysteine, the condensation of the latter with 
serine to form cystathionine, and the cleavage 
of the latter to cysteine and homoserine. A dual 
pathway for proline synthesis has been indicated, 
one involving glutamine as the source of the car- 
bon skeleton, and the other involving arginine 
by way of ornithine. 

An intriguing recent observation has been the 
finding that a number of factors which are rigor- 
ously required by the cells for survival and 
growth can in fact be synthesized. Their nutri- 
tional requirement reflects the fact that they are 
lost from the cellular pool to the medium at rates 
which exceed the biosynthetic capacity of the 
cell; and with a sufficiently high cell population 
density, when the loss to the medium per cell is 
sufficiently reduced, the supposedly essential 
growth factors are in fact not required for sur- 
vival. 

In these cell cultures, unlike bacteria, the bio- 



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129 



synthesis of amino acids is apparently not in- 
hibited by the product of the reaction; and this 
mechanism of growth control is apparently not 
operative. Studies are in progress as to whether 
enzyme repression or feedback inhibition are ef- 
fective controls in the biosynthesis of pyrimidines. 
A quite different control mechanism is perhaps 
indicated by the demonstration of a growth in- 
hibitor in the supernatant medium of heavy cul- 
tures. The chemical nature of that inhibitor is 
under continuing study. 

Studies on the mechanism of resistance to 
2-deoxyglucose (2DG) have shown the presence 
in the resistant variants of compounds which 
inhibit the phosphorylation of 2DG to the meta- 
bolically active inhibitor, 2DG-phosphate. The 
relationship of that inhibitor to the observed 
resistance is under continuing study. 

Viral Synthesis 

A number of important new observations have 
been made with respect to the mechanisms of 
viral synthesis. The puzzling wide disparity be- 
tween the number of physical particles in viral 
suspensions, and the number of plaque-forming 
units, i.e. particles capable of initiating infection 
in susceptible cells, has been partially resolved 
with the demonstration that after the viral par- 
ticle has been absorbed by the cell, it may undergo 
several alternative fates. A large proportion are 
rapidly eluted into the medium, essentially intact 
but no longer infectious, presumably reflecting a 
minor alteration in the protein coat. Some par- 
ticles remain unchanged within the cell. Others 
are degraded intracellularly, in that the nucleic 
acid is exposed and becomes susceptible to intra- 
cellular ribonuclease. Only a small fraction of 
the absorbed viral particles are stripped of their 
protein and initiate infection. 

In the case of poliovirus in the HeLa cell, 
although the viral protein and RNA are syn- 
thesized concomitantly, a partial dissociation has 
been achieved with appropriate inhibitors of pro- 
tein synthesis, which completely block the forma- 
tion of mature virus, but not of infectious RNA. 
This is of particular importance in relation to 
the supposedly obligatory relationship between 
protein and RNA synthesis in growing cells. Of 
interest also is the fact that metabolic inhibitors 
which effectively block the synthesis of cellular 



DNA and of DNA viruses have no effect on the 
formation of poliovirus. It would therefore ap- 
pear that poliovirus RNA may be used directly 
as a template for the formation of virus, without 
the necessity for intervening DNA synthesis. 

In contrast to the situation with poliovirus, in 
the case of vaccinia, there was a marked lag be- 
tween the formation of the viral nucleic acid 
(DNA) and that of the mature virus. 

Galactosemia Cell Cultures 

An exciting new development has been the suc- 
cessful cultivation from patients with a heredi- 
tary metabolic disease (galactosemia) of cells 
which in culture demonstrate the metabolic de- 
fect characteristic of the disease. This suggests 
an entirely new experimental approach to prob- 
lems of human genetics. 



LABORATORY OF GERMFREE 
ANIMAL RESEARCH 

Animal Studies 

A series of observations has been made on the 
behavior of Entamoeba histolytica in the germ- 
free host. In earlier studies with standardized 
techniques, amoebic lesions were not produced in 
the germfree animal following inoculation. In 
fact, the parasite failed to live in the intestine 
beyond five days. Recent changes have been 
made in the manner of rearing and handling the 
amoebae in vitro prior to inoculation which 
seemed to result in more vigorous organisms. The 
latter have produced lesions in the absence of 
bacteria, although the type and severity are still 
not typical of those encountered with a bacterial 
associate. Thus, it would appear that the latter 
is not the only determinant of the course and the 
pathogenesis of the infection. 

Some studies are preparatory to an analysis of 
the nature of the nutritional effects observed in 
certain parasitisms. For instance, the intestinal 
mouse parasite, Nematospiroides dubius, does not 
require a bacterial flora to develop from an infec- 
tive larva to the adult form in the host; but ap- 
parently, it does require a flora to develop from 
the egg to infective larva. Another observation 



130 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



is that the sex of the host, although noted by sev- 
eral workers to be a factor in the outcome of 
infection by this parasite in conventional (con- 
taminated) animals, does not appear to be a fac- 
tor in the germfree host. 

Guinea Pigs 

In studies on the growth and biology of germ- 
free guinea pigs, a staff investigator has obtained 
several advanced pregnancies in animals main- 
tained on irradiated diets, although no fetus was 
carried to term. It is to be recalled that germ- 
free guinea pigs have not yet been bred with 
success. The importance of the intestinal flora 
to this species was pointed up by the finding that 
conventional (contaminated) guinea pigs repro- 
duced normally on this same irradiated diet. 

In a collaborative project with an investigator 
at the University of Pennsylvania, it has also 
been shown that the use of large dosages of a 
cathartic, or the application of tourniquet shock, 
increased the number of red cells of germfree 
chickens coated with human B-like antigens fol- 
lowing monoinfection with Escherichia coli 88 . 
These studies are providing information on the 
manner in which red cells of one type may ac- 
quire antigenic characteristics of other cell types, 
especially B. 

Mouse Colony 

The germfree mouse colony has been undergo- 
ing an intensive serologic study including an 
assay for the presence of certain so-called "natu- 
ral antibodies" against a variety of bacteria. 
Such antibodies or antibodylike reactivities for 
organisms like Staphylococcus, E. coli and Sal- 
monella typhosa have been found to occur in a 
variety of uninoculated conventional animals and 
are presumed to originate from encounters with 
the viable organisms or related antigens. Ani- 
mals which have lived for many generations free 
from contact with live bacteria are almost the 
sine qua non for establishing finally the validity 
of these ideas. Studies thus far, with the Com- 
municable Disease Center and investigators in 
the National Cancer Institute, have shown that 
germfree mice 2-3 months of age were free from 
antibodylike reactivity toward Staphylococcus 
and E. coli antigens. Reactivity, however, toward 



S. typhosa was obtained in several instances, al- 
though no evidence of the presence of the latter 
was found in the germfree colony. Thus, this 
finding strengthens sporadic reports that non- 
bacterial substances (perhaps in this case dietary 
components) can cause "cross" reactions with this 
organism. 

Tumors 

The germfree animal colony and a conventional 
colony derived from the same stock now has ex- 
isted for approximately 2 years. Some of our 
exbreeders, are one to two years of age. When- 
ever a germfree or conventional animal not on an 
experiment dies, especially if it is 6 months or 
more of age, it is examined thoroughly for gross 
evidence of malformations or tumors. Among 
approximately 50 such animals so-called sponta- 
neous lung tumors have occurred in some of the 
germfree as well as the conventional mice. While 
the numbers of animals are obviously small, the 
incidence has been markedly higher, thus far, 
among the conventional animals (those exposed 
to external contamination) than among the germ- 
free. This seems to be particularly true among 
animals 6 to 12 months of age. 



LABORATORY OF PARASITE 
CHEMOTHERAPY 

This country's commitment of $38 million in 
fiscal year 1961 toward a program of world-wide 
malaria eradication and the long-term interest in 
malaria by most of the senior staff resulted in a 
research effort, during the past year, largely di- 
rected toward problems in that field. Special 
emphasis was given to the study of simian ma- 
laria in man and in monkeys because malaria in 
simians might be a real deterrent to the eradica- 
tion program. Clinical facilities for volunteers 
at the Atlanta Penitentiary were enlarged and 
the staff increased. A laboratory was established 
at Kuala Lumpur, Malaya, in cooperation with 
the Malaya Institute of Medical Research and the 
United States Medical Research Unit. Studies 
on several aspects of the simian-human-malaria 
problem have been in progress there since mid- 
August. 

As a result of the above development, it was 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



131 



decided to move the Section on Cytology, now 
located at Memphis, to Chamblee, Georgia, early 
in 1961. This arrangement will bring the simian 
hosts closer to the human volunteers at the peni- 
tentiary, and the insectary maintained by the 
Section will be geared to accommodate the work 
at Chamblee and at the prison. 

Malaria — Human 

Plasmodium, falciparum (McLendon strain) : 
Chloroquine (300 mg, base) and primaquine (45 
mg, base) given together beginning three days 
after mosquito bites and weekly thereafter for a 
total of eight doses, resulted in suppressive cure 
in 5/5 subjects. Controls were positive 11 to 15 
days after infection. After two days of para- 
sitemia, each control was given the above drug 
combination which was repeated weekly for a 
total of three doses. Parasites were removed 
promptly and cure was obtained based on no 
evidence of infection during 227 days of obser- 
vation. 

Primaquine, at daily doses of 0.75 mg, had 
some sporontocidal effect upon Plasmodium falci- 
parum gametocytes but none against those of 
P. vivax (one case). Therapeutic doses (1.4 gm 
in three days) of amodiaquine had no sporonto- 
cidal effect against gametocytes of P. falciparum 
(one case). The effect referred to is against the 
development of the malaria parasites in the mos- 
quito. 

A strain of Plasmodium falciparum from Co- 
lombia, South America, was found to be resistant 
to chloroquine. This finding is of utmost impor- 
tance in terms of malaria eradication. 

Plasmodium vivax (Chesson strain) : A drug 
combination of primaquine (45 mg) and pyri- 
methamine (50 mg) given weekly beginning 
seven days after mosquito bite and continuing 
for a total of four doses, gave suppressive-cure 
in 4/5 subjects; the other subject developed a 
patent infection 240 days after infection. Pyri- 
methamine (50 mg) given alone, as above, pro- 
duced suppressive-cure in 1/4 subjects; the other 
three came down on days 82, 83 and 84. Five 
controls all came down 12 to 13 days after in- 
fection. 

The Russian 8-aminoquinoline, quinocide, was 
compared with primaquine and found to be dis- 
tinctly inferior as a curative drug against early 
and late primary attacks of Chesson vivax ma- 



laria particularly from the standpoint of the 
occurrence of second and third relapses. 

Another 8-aminoquinoline, Win 5037, was stud- 
ied in five subjects. Toxic effects and failure to 
cure made further investigation unwarranted. 

Plasmodium malariae : The results of a 14-year 
study of the biology of Plasmodium malariae 
were drawn together for publication. The high- 
est infectivity for mosquitoes occurred during the 
eighth to tenth weeks of the primary attack. 
Although the infection rate of mosquitoes was 
ordinarily low, the relatively long period during 
which mosquitoes could be infected may explain 
the persistence of P. malariae in nature. The 
ability of the symptom-free malarious patient to 
infect mosquitoes at a rate similar to that of the 
symptomatic patient makes eradication difficult. 

Malaria — Simian 

Plasmodium cynomolgi bastianellii: In early 
May, two accidental sporozoite-induced infections 
with Plasmodium cynomolgi bastianellii occurred 
at our Memphis Laboratory. This happening was 
of signal importance because it showed that 
simian malaria, contrary to the generally held 
opinion, was infectious to man. In that light, 
full scale study of human infections was under- 
taken at our Atlanta Penitentiary installation. 

Two infections were induced in inmate volun- 
teers by inoculation of infected blood obtained 
from one of the accidental sporozoite-induced in- 
fections in man. Twenty inmate volunteers were 
infected by bites of Anopheles quadrimaculatus 
or Anopheles freebomi which had fed on infected 
monkeys. The prepatent period ranged from 14 
to 29 days and the parasite density ranged from 
5 to 500/cmm. The most constant symptom was 
headache and the most significant signs were 
fever, splenomegaly and hepatomegaly. Infec- 
tions were allowed to run their course, generally 
without treatment. 

Anopheles freebomi were infected from two 
patients but attempts to infect volunteers by their 
bites have yielded equivocal results. The finding 
that P. c. bastianellii will grow consistently and 
produce clinical illness in man suggested the 
possibility that malaria is a zoonotic disease, that 
is, a disease which man can acquire from animals 
with which he is associated. Whether or not such 
transfer occurs in nature is not yet determined, 
but should it occur, it would be of greatest sig- 



132 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



nificance to the world-wide malaria eradication 
program. 

Plasmodium cynomolgi cynomolgi: Eleven in- 
mate volunteers were bitten by Anopheles free- 
horni infected with P. c. cynomolgi on 8 Septem- 
ber, and to date (14 December) three have 
exhibited evidence of infection (i.e., fever). 
Parasitemia has been demonstrated in only one, 
on the 58th day after mosquito bites. These re- 
sults show that this strain infects man far less 
readily than P. c. bastianellii. 

Field Studies in Malaya 

Three staff members, Drs. Eyles, Dobrovolny, 
and Mr. Clinton S. Smith, were detailed to 
Malaya during the year where they engaged in 
the study of simian and human malaria in co- 
operation with the Malayan Institute for Medical 
Research and the U.S. Army Medical Research 
Unit at Kuala Lumpur. 

The epidemiology of monkey malarias is being 
studied and the feeding habits of some of the 
Anopheles determined. By injection of unin- 
fected monkeys with sporozoites from natural 
infections, it was determined that Anopheles 
hackeri is a natural vector of Plasmodium know- 
lesi. This is a most important discovery, espe- 
cially since the vector of this parasite has been 
sought for repeatedly during the last 25 years. 

Studies of malaria in aborigenes associated 
with monkeys have been made. Blood passed 
from aborigenes to monkeys have thus far pro- 
duced no patent infection in the monkeys. 

EE Stages and Drug Action 

Studies were continued on the direct effect of 
drugs on the exoerythrocytic stages of primate 
malaria. When sulfonamides were used with 
pyrimethamine to exploit the possible synergism 
of the two drugs, monkeys developed parasitemia 
30 to 40 days after inoculation with sporozoites 
even though all parasites observed in liver biop- 
sies were damaged. The curative efficacy of 
quinocide, the Russian drug, was compared with 
primaquine. Even when administered at twice 
the dosage used with primaquine, quinocide was 
less effective. Chloroquine had no observable 
effect upon the liver forms of Plasmodium cyno- 
molai. Young parasites appeared in the blood 



in large numbers on the 8th, 16th, and 24th day 
indicating the existence of secondary exoerythro- 
cytic generations. 

Insect Tissue Culture 

Blood cells from caterpillars and cells of the 
ovariole sheath of several species of moth pupae 
have been cultivated in several different media. 
The virus of St. Louis encephalitis has been main- 
tained in cultures of hemocytes from larvae of the 
catalpa sphinx for ten days. Oocysts of Plasmo- 
dium gallinaceum attached to the midgut of 
Aedes aegypti have shown growth in vitro and 
sporozoites have been produced. 

Biochemical Studies 

It was shown that mosquitoes infected with 
malaria have higher levels of ribonucleic acid 
than uninfected mosquitoes. Chromatographi- 
cally, the acid-hydrolysate of ribonucleic acid 
from a pyrimethamine-resistant strain of Plas- 
modium falciparum differs from the acid-hy- 
drolysate of ribonucleic acid from a pyrimeth- 
amine-susceptible strain. Bephenium hydroxy- 
naphthoate inhibited glutamic acid transaminase 
of Nippostrongylus muris. Bephenium chloride 
and quinacrine reduced the rate of glucose ab- 
sorption by the tapeworm Uymenolepis diminuta 
but low concentrations of dithiazanine iodide 
stimulated glucose absorption by this cestode. 

Intestinal Parasites 

Epidemiological studies on the inmates of a 
mental institution show a high persistence of 
Trichuris and hookworm for six years, with an 
apparent decrease in Strongyloides. To test 
dithiazanine and tetrachlorethylene, alone and in 
combination, heavily parasitized mental patients 
were given the drugs for about one year. A large 
number of worms were removed but the cure rate 
was low and transmission was not stopped. 
Bephenium hydroxynaphthoate and bephenium 
chloride were used with good results against 
hookworm, Ascaris and Trichuris. 

Schistosomiasis 

The activity of griseofulvin observed in mice 
infected with Schistosoma mansoni was not well 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



133 



developed in hamsters or monkeys. A series of 
tetracycline analogues which show an affinity for 
microfilaria did not combine with schistosomes 
and were without activity. One of these ana- 
logues was significantly more active against mi- 
crofilariae of Diroflaria immitis than tetracycline. 
In many tests, the efficacy of stibophen 
(Fuadin) therapy on mature Schistosoma man- 
soni infections in mice was increased up to 16 
times by feeding a balanced semi-synthetic diet. 
The toxicity of the drug was not similarly in- 
creased. The enhancement of curative action by 
the purified semi-synthetic diet was thought to be 
due to the absence of, as yet unidentified, inor- 
ganic salt (s) that interfere with drug activity. It 
was found in mice fed on the purified semi-syn- 
thetic diet that higher blood levels of the drug 
were maintained for a longer period than when 
the same amount of Fuadin was injected into 
mice fed on the commercial pellet diet, suggesting 
that the increased cure-rate was due to higher 
blood drug level. Similar drug advantage was 
observed in mice given tartar emetic while on 
the purified diet. 



LABORATORY OF PARASITIC DISEASES 

This Laboratory continues to emphasize fun- 
damental studies on parasites and parasitic dis- 
eases. No important changes in the program 
were instituted during the year. The program 
of the laboratory is well diversified considering 
the size of the staff and the competencies of the 
various staff members cover a large proportion 
of the field of parasitology. 

Although the emphasis is on basic studies, this 
does not imply a narrow viewpoint and the labo- 
ratory is well aware of the many practical prob- 
lems parasitic diseases create throughout the 
world. The laboratory is often called upon for 
help and advice concerning prevention and con- 
trol of parasitic infections and so must maintain 
competence, and a reputation for competence, to 
deal not only with basic problems of parasitism 
but also problems of prevention and control of 
parasitic diseases. Therefore, the laboratory con- 
tinues to carry on a variety of activities which 
help it maintain its international reputation and 
increase its capacity to cope with problems of 
parasitism. Such activity also returns benefits in 



the forms of ideas for laboratory research and 
clues which may explain puzzling laboratory 
findings. 

Toxoplasmosis 

Studies on toxoplasmosis in New Zealand sheep 
have shown that the prevalence is high. New 
information has been obtained concerning the 
distribution of the organisms in the tissues and 
their persistence there. After inoculation the 
distribution of the parasite in tissues is erratic 
and the parasites rapidly disappear from tissues 
other than the muscle and placenta. Since re- 
sidual infection occurs in muscle, mutton may 
serve as a source of human infection. Congenital 
infection with Toxoplasma is an important medi- 
cal problem, therefore it is of special interest that 
the sheep studies have indicated that inoculation 
of sheep 60 days before pregnancy did not result 
in congenital infection or abortion but inocula- 
tion at 30 days pregnancy caused abortion or 
foetal death with absorption. Infection at 90 
days pregnancy was less likely to be dangerous 
to the foetus. 

The status of resistance or immunity to Toxo- 
plasma continues to be puzzling, since living 
organisms fail to protect completely animals 
against challenge, especially when the challenge 
is great, and because low grade parasitemia may 
persist for months in mice and rabbits in the 
presence of high serum antibody levels. The ob- 
servation that cysts of Toxoplasma probably form 
in tissue cultures provides a new opportunity to 
study the manner of cyst formation and the fac- 
tors that lead to cyst formation. 

Amoebiasis 

The work on the preservation of living Enta- 
moeba histolytica and other protozoa has practi- 
cal significance since success would permit reten- 
tion of strains without continuous sub-culturing. 
This is a relatively new field and techniques are 
still evolving. The work so far has shown that 
this approach is feasible since four species have 
been frozen and stored for periods ranging from 
one to four months depending on the species in- 
volved. E. histolytica has been kept at -197° C. 
for 24 hours, suggesting that almost indefinite 
storage at this temperature may eventually be 
achieved. 



134 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Laboratory culture of E. histolytica continues 
to receive attention since it is so important to 
learn more concerning its nutritional requirements 
and its pathogenicity in the absence of other 
organisms. It is noteworthy that satisfactory 
axenic culture of this species has been achieved 
for the first time. The protozoa are cultured in 
a complex diphasic medium containing no cells 
but including chick embryo extract. 

The substitution of a species of Crithidia for 
Trypanosoma cruzi in cultures of E. histolytica 
provides a more economical and rapid way of 
producing large cultures of the amoeba. Dem- 
onstration of the value of the Coulter Counter 
for the enumeration of protozoa in suspension 
adds a valuable tool for quantitative work and 
suggests this method may be applicable for count- 
ing other organisms of similar size such as tissue 
culture cells. 

Parasitic Infections in Germfree Animals 

The use of germfree animals in worm-parasite 
studies continues to reveal the value of this tool 
and adds to our knowledge of the peculiar nature 
of the germfree state. The technique seems to 
be particularly useful for studying conditions 
that influence natural resistance and nutritional 
relationships of parasite and host. For example, 
it was found that the roundworm, Nematospi- 
roides dubius, develops as well in germfree as in 
conventional mice but while in conventional mice 
the worm recovery is much higher from the male 
animals, the recovery from germfree mice is the 
same for both host sexes. The cause of the dif- 
ference is unknown. Also, it has been shown that 
the feces of germfree mice do not support devel- 
opment of N. dubius larvae and that bacteria in 
the feces provide important factors for larval 
development. There was further evidence that 
the alteration in levels of serum protein compo- 
nents in germfree animals is due to dietary 
factors. 

Sterile Culture of Worms 

Studies on the sterile culture of worms con- 
tinues to produce fundamental information on 
the nutritional requirements of the parasites and 
brings closer the day when we can use the axenic 
animals for immunologic and therapeutic studies. 



Survival studies using relatively advanced larvae 
of Nippostrongylus muris has produced important 
results. The intent has been to try, by addition 
of elements to the medium, to induce the larvae 
to reach the adult stage. Starting with a salt 
mixture, dextrose was added until the optimal 
level was reached. Then casein was added and 
survival time rose to 11 days, but there was not 
development of the larvae. Addition of a yeast 
extract to this mixture not only increased sur- 
vival but permitted growth to the adult stage. 
Thus, a much more simple medium than used 
before has been evolved and the achievement of 
a denned medium for culture of N. muris adults 
is much closer. A similar approach is being used 
in attempts to culture microfilariae of Diroflaria 
immitis. 

Nutrition and Schistosomiasis 

Although the study of the relation of nutrition 
to schistosomiasis in Puerto Rico is still incom- 
plete, it appears that enrichment of the diet does 
not affect the number of eggs passed in the feces. 
However, it is interesting to note that the en- 
riched diet did cause a loss of hookworms and 
whipworms from the intestine. This has a bear- 
ing on the problem of the existence of hookworm 
infection without hookworm disease. In labora- 
tory studies conducted in Bethesda the enhanced 
efficacy of stibophen in mice receiving a semi- 
synthetic diet was shown to be due to the absence 
from this diet of as yet unknown inorganic salts. 

Dual Virus and Helminth Infections 

Interaction of two pathogenic organisms in the 
same host has had relatively little attention in 
spite of some very provocative work done in years 
past. A study of simultaneous infection with 
encephalomyocarditis virus and Trichinella spi- 
ralis in rats has produced striking and significant 
results. While the virus alone does not injure 
adult white rats when given intraperitoneally, in 
the presence of Trichinella spiralis infection 
many of the rats are crippled and die. This 
potentiation of virus pathogenicity is not due to 
nonspecific stress but seems to be related to the 
presence of the worms on the muscles. The virus 
can be recovered from the muscle of T. spiralis- 
inf ected rats but not from muscle of rats without 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



135 



T. spiralis. The reason for the influence of the 
worm infection on the activity of the virus is un- 
known. The phenomenon offers an opportunity 
to study some of the fundamental factors in the 
pathogenesis of both the virus and the worm 
parasite. It also provokes the question as to 
what effect this worm infection may have on 
other virus infections. 

Ammonia Toxicity in Mice 

The study of liver damage in relation to am- 
monia toxicity in mice has revealed that low oxy- 
gen in breathed air greatly enhances ammonia 
toxicity. The mechanism of this effect is not 
clear. Though hepatic coma is usually considered 
to be related to ammonia toxicity none of the 
substances which exacerbate hepatic coma in man 
increases ammonia toxicity in mice. In fact, six 
of ten decrease it. Ammonia toxicity in mice was 
greatly reduced by hypothermia and this suggests 
that the same measure may be useful in treating 
hepatic coma in man. Finally, mouse liver dam- 
age was induced in eight different ways but none 
caused any change in the animal's response to 
intravenous ammonia.- Thus, though high blood 
ammonia levels seem to be related to liver dam- 
age, the causal relationships are by no means 
clear. 

Helminths 

Fundamental physiological studies have focused 
on the calcareous corpuscles of tapeworms and on 
the phospholipids of tapeworms. The calcareous 
corpuscles are amorphous but, on heating, dolo- 
mite, brucite or apatite may be formed. Electron 
microscope pictures of corpuscles heated with 
KOH reveal the presence of well-formed crystals. 
The glycerol containing phospholipids of Taenia 
taeniaeformis are about half lecithid and half 
cephalin. Sphingomyelin is present and more 
than one cephalin is known to occur in the larvae 
of this tapeworm. Hexose-containing phospho- 
lipids occur in both larvae and adults. 

Study of the mechanism of energy metabolism 
of sub-cellular elements has dealt, among other 
things, with the mechanism by which mitochon- 
dria which are depleted of high-energy phosphate 
intermediates are stimulated to oxidize substrates 
when ATP is added. This is a complex, though 



fundamental, bioenergetic system for which a 
better understanding is needed. Addition of 
ATP not only restored succinate oxidation but 
also caused reduction of intra-mitochondrial 
DPN. The succinate oxidation involves an en- 
ergy-requiring reaction and this energy is appar- 
ently added at one site in the respiratory chain 
and used at another for reducing pyridine nu- 
cleotide. 



LABORATORY OF BIOLOGY OF VIRUSES 

The basic objectives of this laboratory continue 
to be the same as last year. It is obvious from 
this annual report that four out of five units have 
projects with the same general objective — inves- 
tigation of mechanisms and localization of animal 
virus synthesis within the infected cell. Each of 
these units is also interested in the infectious 
nucleic acid of viruses. In view of the complexity 
of this problem and the important implications 
of any information that is obtained, this "du- 
plication" is quite justified. Actually, it is not 
duplication since different approaches are used 
and different virus-cell systems are studied. 

The electron microscope has been installed and 
is now used not only by the Biophysical Unit 
but also by other units of our laboratory and by 
units of the Laboratory of Infectious Diseases, 
With studies on the structure of viruses and a 
project concerned with the genetics of animal 
viruses added to the biochemical and biological 
studies, there is now fairly complete coverage of 
the important facets of basic virus biology. 

Intracellular Location of Poliovirus 

By use of radioautographs and staining with 
fluorescein-tagged antiviral antibody, the intra- 
cellular location of poliovirus antigen — presuma- 
bly viral protein — during the cycle of virus mul- 
tiplication has been determined. Demonstrable 
antigen first appeared one hour after infection 
and was diffusely distributed through the cyto- 
plasm. At three hours, just before the appearance 
of new virus, it was present throughout the nu- 
cleus with a tendency to be concentrated around 
the periphery of the nucleolus. At five to seven 
hours, particulate accumulation of antigen in the 
cytoplasm was noted. Incorporation of radioac- 



136 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tive-tagged amino acid into cell protein ceased 
shortly after the start of infection, whereas in- 
corporation of thymidine into RNA continued 
until after three hours and tended to localize in 
the nucleoli. 

Mutants of EMC Virus 

Plaque type mutants of EMC virus have been 
found, segregated and characterized. The sta- 
bility of the mutants has been determined and 
the plaque type shown to be a function of the 
viral ENA. It has been shown that the difference 
in the size of the plaques formed by these mutants 
is brought out by an inhibitor present in the agar 
overlay used on the plaque plates. This inhibitor 
resides in the agaropectin fraction of the agar 
and can be separated from the agarose fraction 
which then permits both plaque type mutants to 
form similar sized plaques. 

Polyoma Virus 

By the use of a serum protection test in new- 
born hamsters, evidence was found that polyoma 
virus transforms normal cells to tumor cells 
quickly and directly without extensive virus mul- 
tiplication being necessary. Furthermore, no 
evidence could be found to suggest a lysogenic 
relationship of virus to tumor cell. All attempts 
to show the presence of infectious or masked 
virus or of virus antigen in transplantable poly- 
oma-induced tumors have been negative. It ap- 
pears that once the virus initiates the tumor it 
is no longer required for tumor growth and 
maintenance. 

Tetracycline Fluorescence 

The discovery has been made that when the 
antibiotic tetracycline stains tissues in such a way 
that they fluoresce under UV light, this fluores- 
cence is localized in the mitochondria of the cells. 
This makes a convenient vital stain of these sub- 
cellular elements for further studies. There ap- 
pears to be some similar localization of the anti- 
biotic fluorescence in certain bacteria. 

TMV Model 

A complex model of tobacco mosaic virus has 
been constructed on theoretical grounds, and on 



checking this model against known biochemical 
and biophysical properties of the virus a remark- 
able consistency is found. Certain refinements of 
electron microscopic technics have produced pho- 
tographs of this virus which reveal previously 
not seen fine structure also consistent with the 
theoretical model. 



LABORATORY OF IMMUNOLOGY 

Since the activation of the Laboratory of Im- 
munology in 1957, the program has been con- 
cerned, principally, with basic research. How- 
ever, for some time an important need has been 
felt for the initiation of clinical studies in im- 
munology and allergy. In September 1960 the 
Clinical Immunology Section was activated and 
as space permits, will be expanded and will work 
in close collaboration with the Laboratory of 
Clinical Investigation on clinical studies involv- 
ing immunological aspects of such diseases as 
lupus erythematosus, nephritis, and chronic thy- 
roiditis, in which an auto-immune basis is sus- 
pected. 

Allergic Thyroiditis 

Experimental allergic thyroiditis was produced 
in Strain 13, inbred, histocompatible guinea pigs 
by immunization with a single dose of guinea pig 
thyroid extract in complete Freund's adjuvant. 
Thyroiditis developed as early as five days after 
immunization, was present in all animals at 16 
days, and by seven weeks was consistently pres- 
ent and generally severe. Delayed skin test hy- 
persensitivity was found as early as five days 
after immunization in nearly all animals, and 
was present in all animals with thyroiditis at 
seven weeks. At seven weeks after immunization, 
anti-thyroid antibodies were present, and anti- 
body titres correlated with the presence and 
degree of thyroiditis. This correlation was not 
found at certain other times after immunization. 
The presence of delayed hypersensitivity was 
correlated with experimental allergic thyroiditis, 
while the presence of circulating antibody did not 
correlate with thyroiditis. These observations 
constitute the earliest production of experimental 
allergic thyroiditis and the most severe disease 
at the time intervals studied. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



137 



House Dust Allergens 

Studies on the chemical and physical properties 
of house dust extracts that are used clinically for 
the diagnosis and treatment of house dust allergy 
have been studied to identify the components re- 
sponsible for the specific skin reactions produced 
in house dust sensitive individuals. It has been 
found that the house dust extracts consist of a 
heterogeneous mixture of acidic polysaccharides. 
The heterogeneity has been demonstrated by elec- 
trophoretic and ultracentrifuge sedimentation 
analysis and also by the multiplicity of cross re- 
actions obtained with antisera to the various 
pneumococcal polysaccharides. The chemical com- 
position of the various fractions has been shown 
to be roughly 5 to 20 percent polypeptide and 
80 to 95 percent polysaccharide, containing about 
equal amounts of uronic acid (probably glucu- 
ronic acid), D-glucose, D-galactose, D-mannose 
with lesser amounts of L-rhamnose and L-arabi- 
nose. 



Genetics of Gamma Globulin 

Agar-gel immunochemical analysis of sera from 
rabbit litters, with precipitating antibodies pre- 
pared in rabbits, has shown that seven antigenic 
determinants of the gamma globulins are geneti- 
cally controlled by at least two gene loci with 
each specificity exhibited when the appropriate 
allele is present. Since the gamma globulins are 
soluble proteins which have properties of both an 
antigen and an antibody, they should be subject 
to quantitative estimation and cytological locali- 
zation. This immunogenetic system, therefore, 
may be uniquely suited for the study of certain 
basic problems in genetics, embryology, immunol- 
ogy and protein chemistry. 

In other studies, antibodies to human serum 
proteins were prepared in monkeys since this 
animal, being a closely related species, might be 
more discriminating for minor antigenic differ- 
ences than a distantly related species. Three 
"slow" gamma globulins were found, instead of 
the one usually detected with horse or rabbit 
antibodies. Two of these were shown to be re- 
lated to myeloma proteins. The quantitative esti- 
mation of these gamma globulins in serum should 
be helpful in the early diagnosis and study of 
diseases, such as multiple myeloma, which in- 



volve qualitative and quantitative changes in the 
gamma globulins. 

Hypersensitivity 

The genetically distinct guinea pigs of inbred 
Strains 2 and 13 have proved to be a very impor- 
tant immunological tool. After studies estab- 
lished the fact of skin compatibility in the two 
strains, experiments were conducted to transfer 
cells with a measurable biological activity. Trans- 
fers of tuberculin sensitivity were undertaken by 
the intraperitoneal injection of living lymphoid 
cells from compatible donors. The almost quan- 
titative transfers between inbred guinea pigs were 
a reflection of the continued viability of the 
active cells in the recipients. 

Two models are being developed to study the 
mechanisms of immediate and delayed hypersen- 
sitivity in the inbred guinea pigs; protracted 
anaphylactic shock and, the massive local hemor- 
rhagic reaction, respectively. It has been shown 
that there are differences in susceptibility to hy- 
persensitivity reactions. Strain 2 guinea pigs 
were more resistant to death by bronchospasm 
and tended toward a protracted syndrome in 
anaphylactic shock. Both Strain 2 and 13 guinea 
pigs required more mycobacteria than did ran- 
dom-bred Hartley guinea pigs for inducing "de- 
layed" sensitivity to egg albumin, using Freund's 
adjuvant. 

Human Serum Auto Antibodies 

Fractions of human serum separated by anion- 
exchange cellulose column chromatography were 
studied by Immunoelectrophoresis. The condi- 
tions for elution of eighteen immunologically dis- 
tinguishable human serum proteins from the 
columns were determined. Gamma globulin ob- 
tained under the appropriate conditions by this 
method was found to be pure ; rabbits immunized 
with this fraction made antibodies to none of the 
other serum proteins. By the use of anion-ex- 
change cellulose columns, it has been found pos- 
sible to separate the 7S from the 18-19S antibody 
activities in sera of patients with thyroiditis and 
lupus erythematosus. Initial results indicate that 
the addition of immunoelectrophoretic characteri- 
zation of these and other sera will be extremely 
helpful in our aim of characterizing the antibody 
activities found in human serum. 



138 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Fluorescent 4ntibody Staining of 
Malaria Parasites 

The fluorescent antibody staining of the human 
malaria parasite, Plasmodium vivax, has been 
recorded ' for the first time. A globulin fraction 
of convalescent serum from a patient having a 
long-standing infection with P. vivax was labeled 
and the fluorescent antibody applied to thin blood 
films containing the parasite. The organism was 
visible by virtue of its specific immunofluores- 
cence. Fluorescent antibody studies were con- 
ducted on P. cynomolgi oastianellii, the monkey 
malaria parasite which, recently, has been shown 
to be transmissible to man. Considerable mor- 
phological detail was observed at fluorescence. 
Preliminary studies on the staining reactions, as 
based on degrees of fluorescence, indicate that 
P. vivax and P. cynomolgi oastianellii parasites 
may have common antigens and that the two 
species may be closely related 



LABORATORY OF INFECTIOUS DISEASES 

In 1960 the Virus and Rickettsial and Epidemi- 
ology: Sections of this laboratory continued in- 
tegrated and comprehensive efforts to define the 
importance of virus infections in disease. Field 
investigations of human and animal virus infec- 
tions were made possible through collaborations 
with a number of other organizations, including 
the Bureau of Medicine, USN ; the District of 
Columbia Children's Hospital Research Founda- 
tion; the District of Columbia Welfare Depart- 
ment; the New York City Health Department; 
the National Cancer Institute; the National In- 
stitute of Allergy and Infectious Diseases; the 
Laboratory of Clinical Investigation, NIAID; 
and in Paris, France the Laboratoire des Virus, 
Hopital Saint- Vincent-de-Paul ; and Le Centre 
Claude-Bernard de l'Hopital Saint Louis. 

Natural events and opportunities afforded by 
our collaborators shaped the course of most field 
studies. Technical breakthroughs in the labora- 
tory made it possible to take fuller advantage of 
these opportunities to study natural disease and 
thus acquire not only new information about 
specific virus infections, but also to move nearer 
our ultimate goal, namely, a clear view of the 
numerous viral causes of human diseases suffi- 



ciently comprehensive to make concerted efforts 
to control them appear feasible and worthwhile. 

Virus Pneumonia 

Pneumonia and other lower respiratory tract 
infections continue to represent major causes of 
death and a large segment, presumed to be viral 
in origin, is still uncontrolled. Until recently it 
was wholly undefined. During 1958 and 1959 
our studies at Children's Hospital and Junior 
Village helped define the relative importance of 
adenoviruses, para-influenza viruses, and influ- 
enza viruses in causing lower respiratory illnesses 
of childhood. The data suggested that as much 
as 40 percent of croup bronchiolitis and pneu- 
monia were explained by these viruses. In 1960, 
using more sensitive methods, we were able to 
explain a much larger percentage of such ill- 
nesses, chiefly because we were now able to assess 
the very significant contributions of respiratory 
syncytial virus (RS) to the respiratory disease 
problem. Early in the year large outbreaks of 
RS virus were intensively studied both at Chil- 
dren's Hospital and Junior Village. Over 80 
strains of RS virus were isolated from children 
with pneumonia and 60 percent with bronchiolitis 
yielded RS virus, whereas virus was recovered 
from less than one percent of comparable control 
patients without respiratory illness. 

Retrospective analyses of serologic surveys of 
respiratory illnesses in Children's Hospital since 
1957 suggested that perhaps 20 percent of all 
lower respiratory illnesses observed during the 
last three years was due to RS virus. Thus, con- 
sidering the contributions of adenoviruses, para- 
influenza viruses, influenza viruses, and "PAP" 
virus it now appears that 50 to 60 percent of the 
more severe respiratory illnesses of young chil- 
dren can now be explained and, hopefully, con- 
trolled. Except for influenza virus (which con- 
tributed probably less than 5 percent of the total) , 
the LID respiratory virus unit personnel played 
key roles in the discovery of the first representa- 
tives of each of the other virus groups — adeno- 
virus, para-influenza, and RS. Delineation of 
still undefined viral causes of the respiratory dis- 
ease syndrome represents the major challenge to 
Tespiratory disease investigators for 1961. 

During 1960 several experimental but commer- 
cially prepared killed vaccines containing various 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



139 



combinations of adenoviruses (6 types), para- 
influenza viruses (3 types), and Coxsackie B 
viruses (5 types) were tested in Junior Village. 
The evidence suggests that while modestly anti- 
genic, the vaccines had insufficient potency to be 
regarded as satisfactory for larger scale studies. 

Primary Atypical Pneumonia 

The etiologic role of PAP (Eaton's virus) in 
primary atypical pneumonia suggested earlier by 
Eaton and Liu, was finally fully established in 
1960. In cooperation with the Bureau of Medi- 
cine, USN, the continuing "epidemic" of virus 
pneumonia in Marine recruits at Parris Island 
was studied in several ways. Serological studies 
showed that 51 percent of 530 pneumonia cases 
had antibody rises to PAP virus; only 6 percent 
revealed contemporary infection with adenovi- 
ruses. Serologic studies of infection showed PAP 
virus to be much more common than disease; 
approximately 30 recruits were infected for each 
case of pneumonia, information vitally important 
to fuller comprehension of the natural history of 
this important virus. 

In 1959 treatment of Parris Island pneumonia 
cases with broad spectrum antibiotics (tetracy- 
clines) appeared to reduce the severity and the 
duration of the Eaton pneumonias. In 1960 the 
efficacy of a new tetracycline drug, demethychlor- 
tetracycline, was tested in a well-controlled double 
blind study including 290 pneumonia patients. 
The drug greatly reduced the severity and dura- 
tion of pneumonitis and fever in those shown to 
have serologic responses to PAP virus. These 
findings, based on accurate laboratory diagnosis, 
fully confirm earlier but controversial reports of 
the efficacy of tetracyclines in atypical pneumo- 
nias. It also adds further support to the impor- 
tance of the Eaton virus as a cause of virus 
pneumonia. 

An additional link in the chain of evidence 
establishing the PAP virus as an important cause 
of pneumonia was achieved recently in collabora- 
tive studies with the Laboratory of Clinical In- 
vestigation, NIAID. Volunteers inoculated intra- 
nasally with PAP virus grown in tissue cultures 
reacted with a wide gamut of respiratory signs 
and symptoms, including pneumonitis character- 
istic of PAP. 



Common Colds and Viruses 



Recent studies have served to clarify and en- 
large existing concepts of the etiology of common 
mild respiratory illnesses in adults. It is now 
quite clear that instead of a few specific closely 
related viruses, numerous viruses belonging to 
different groups each contribute in part to the 
syndrome called the "common cold." Thus the 
newer viruses (adenoviruses, para-influenza vi- 
ruses, respiratory syncytial virus and others) , to- 
gether with older agents (influenza viruses and 
certain bacteria), each contribute only a small 
proportion of the milder respiratory ailments of 
adults. They contribute a larger segment of 
more serious diseases, particularly in children. 
Very recent reports of common cold viruses from 
England, together with the prior reports of agents 
with somewhat similar properties in this country, 
served to focus our attention on these viruses in 
1960. Together with investigators elsewhere, it 
was found that most, if not all, of these agents — 
the British HGP and FEB, the American 2060, 
JH, Coe and PETT viruses which grow selec- 
tively and rather "fussily" in human epithelial 
cell lines, really represent "fastidious" enterovirus 
strains which have (as do almost all Coxsackie 
A's and some ECHO viruses) special growth re- 
quirements. These viruses, as do a number of 
still unclassified agents found in Junior Village 
during the past several years, have properties 
very similar to the Coxsackie A viruses; indeed, 
several have been shown, on the basis of serologic 
markers and/or by suckling mouse pathogenicity, 
to be indistinguishable from Coxsackie A viruses. 



New Serological Test Procedures 

The laboratory section of the Epidemiology 
Section concentrated on the development, appli- 
cation and evaluation of in vitro test procedures 
for the identification of new viruses as well as 
for detecting virus infection as expressed in anti- 
body responses. Thus, using conventional com- 
plement fixation (CF) and newly developed 
hemagglutination inhibition (HI) procedures it 
has been possible for our group to type thousands 
of virus isolates belonging to the adenovirus, 
myxovirus, enterovirus, and reovirus groups. As 
was true during the past several years, LID in 
1960 again described and characterized more new 



140 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



representatives of these viruses than all other 
virus laboratories in the world combined. This 
was made possible during 1960 because each of 
our various virus research units contributed new 
diagnostic techniques. One group developed ad- 
ditional specific HI procedures for identifying 
adenoviruses and adenovirus infections; and for 
reoviruses and enteroviruses as well. Similarly, 
another group developed tissue culture procedures 
for isolating Eaton's PAP virus, while others not 
only discovered several "new" mouse viruses in 
tumor virus study systems, but developed sero- 
logical procedures for recognizing their presence. 

Serologic Reagents 

But the availability of simplified procedures 
are of very little use unless the necessary reagents 
are also available. Although many virus research 
laboratories could do the tests, few laboratories 
are able to produce the necessary reagents. The 
magnitude and cost of producing and certifying 
them promise to continue to exceed any possible 
resources available. This fact has had a very 
depressing effect on research efforts aimed at the 
study of viruses as causes of disease, and serves 
as yet another deterrent to early delineation of 
the common virus diseases as public health prob- 
lems. Consequently, with the help of NINDB 
and Microbiological Associates, LID in 1959 and 
1960 accepted responsibility to develop and evalu- 
ate more than a hundred commercially produced 
virus antigens. LID, of course, has been active 
in the certification of virus prototypes and fur- 
nishes many to the Virus Registry of the Ameri- 
can Type Culture Collection. It is also collabo- 
rating with the Enterovirus and Adenovirus na- 
tional committees in setting up standards for 
large scale production of certified antiserums for 
serotyping and classification of viruses, perhaps 
the highest priority need of all virus laboratories 
concerned with human infection and disease. 

Reference Laboratory for Viruses 

Wholly through the operation of circumstances, 
the Virus Section of LID has become virtually 
the chief (in many instances only) reference 
laboratory for many of the newer viruses, includ- 
ing adenoviruses (about 30 human and several 
animal serotypes), myxoviruses (five new para- 



influenzas occurring in three species), reoviruses 
(three serotypes in four species), many of the 
newer and some older enteroviruses (5-10), sali- 
vary gland viruses (from four species), and new 
mouse viruses (six), the latter frequently found 
in tumor virus study systems. 

Until virus reagents desperately needed for 
many extremely common viruses are made avail- 
able either commercially, through government 
agencies, or both, LID as the sole custodian of 
many of these agents cannot avoid responsibility 
for assisting other excellent virus laboratories to 
identify their viruses, and on a pro-tem basis at 
least for keeping order in the general virus field. 
Unfortunately it has no specific commitment to 
provide such services and even worse, no specific 
budget to cover them, so that the involuntary, 
constantly growing and unavoidable service func- 
tions must be done at the expense of research 
missions. 

However, it must be admitted that the simpler 
virus diagnostic techniques and the availability 
of a complete supply of viral reagents in the 
laboratory (developed out of necessity) facilitate 
not only epidemiologic studies of naturally occur- 
ring virus infection but also enable it to evaluate 
the significance of the data furnished by other 
laboratories who come for technical assistance. 

Cancer Viruses 

Studies of cancer viruses can be subdivided into 
several categories: (a) Laboratory studies of the 
properties of cancer viruses and development of 
laboratory tools for detecting and working with 
them; (b) field studies of the behavior in nature 
of those tumor viruses for which suitable detec- 
tion tests are available; (c) studies of extraneous 
viruses ("background noise") now preventing 
high caliber virologic practice in the study of 
animal tumor viruses and obscuring interpreta- 
tion of nearly all current observations on them; 
and (d) the study of general virus experiences in 
relation to human cancer — the "background noise" 
in the human cancer problem — which must be 
done eventually if the role of viruses in human 
cancer is to be defined. 

The approach to these various interdependent 
studies is based on the following beliefs : ( 1 ) That 
the conventional methods of standard virology 
must be applied to cancer virus research if sig- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



141 



nificant progress is to be made; (2) the study of 
cancer viruses obviously cannot be separated from 
general virology; and (3) that the "biologic 
point of view" rather than attitudes fostered by 
preoccupation with categorical disease, represents 
the best approach to a real understanding of the 
natural history of cancer viruses just as it does 
to other viruses. 

Mouse Polyoma Cancer Virus 

New in vitro survey tools developed during 
1959 (CF, HI, and MAP) were evaluated and 
applied in 1960 in studies of polyoma virus 
growth and excretion, its experimental epidemi- 
ology, and its natural history. This interesting 
and versatile cancer virus causes tumors not only 
in all strains of Mus musculus, but also in ham- 
sters, rats, rabbits, and guinea pigs (Stewart and 
Eddy) . Of equal interest is the fact that it can 
be studied and surveyed with the same facility 
as ordinary viruses, such as influenza and polio- 
viruses. Virus isolation and serologic procedures, 
combined with epizootiologic studies have pro- 
duced the following interesting observations : 

Polyoma virus was found to be widely dissemi- 
nated in mouse colonies nearly everywhere. In- 
fection was found to be more commonly present 
than absent in laboratory strains raised in experi- 
mental or commercial laboratories and in wild 
strains found in city tenements. However, the 
basic ecology or natural cycle appears to exist 
in rural areas — on farms and in feed mills in 
small towns. 

A full year's surveillance of Mus musculus in- 
festation and polyoma infection of crowded tene- 
ments in Harlem revealed that virus infections 
persisted without exception in numerous separate 
foci. Three epidemiologic factors seem most im- 
portant, namely, large mouse populations capable 
of furnishing adequate supplies of young suscep- 
tible mice, the extensive contamination of the 
tenement environment (virus was demonstrated 
in sweepings from areas showing signs of mouse 
activity) , and finally the overcrowding which in- 
sures the continuous and extensive use of com- 
munal nesting areas (also demonstrated to be 
contaminated by virus) . Apartment houses hav- 
ing smaller and less dense mouse infestation were 
generally free of infection and remained so during 
the study. 



Systematic studies of polyoma in rural environ- 
ments were undertaken during the last quarter 
of 1960. However, it appears from preliminary 
data that here may be found the basic natural 
cycle of mouse polyoma. Mus musculus infesta- 
tion and polyoma infection of Mus was found to 
be most intense in feed granaries on the farm and 
in cereal grain storage elevators in mills. As 
many as 30 percent of several hundred mice 
trapped in these environs showed persistent evi- 
dence of polyoma infection, many of them appar- 
ently excreting virus in their urine. The virus 
has been found on cereal grains in the vicinity 
of mouse nesting areas, which appear to be very 
numerous in the granaries so far examined. The 
actual extent of cereal grain contamination by 
mouse excreta containing polyoma and no doubt 
other microbes must still be evaluated; however, 
present evidence suggests that it probably is very 
extensive, if not appalling. 

Since natural infection of wild mice is not lim- 
ited to polyoma virus, but includes a number of 
other viruses known or suspected to infect man 
and domestic animals, the extension of these pre- 
liminary findings will likely prove very inter- 
esting. 



Extraneous Cancer Viruses 

In 1960 the "background noise" problem in 
cancer virus research grew to almost "deafening" 
proportions and, in the opinion of LID virolo- 
gists, constitutes the number one obstacle to in- 
telligent and truly effective research on cancer. 
viruses. 

Nearly every animal tumor virus system cur- 
rently under study was shown to be contaminated 
with extraneous agents and several viruses widely 
proclaimed as "tumor" viruses turned out to be 
fellow traveling ordinary viruses. To list a few 
examples: Friend leukemia was found contami- 
nated with polyoma and mouse adenovirus; 
Gross leukemia by polyoma, K virus and mouse 
adenovirus; Schwartz leukemia with polyoma, 
K virus and mouse adenovirus ; Moloney leukemia 
with mouse hepatitis and mouse reovirus; the 
polyoma itself became contaminated with mouse 
adenovirus, hepatitis and salivary gland viruses. 

LID virologists showed that the "seeds" of the 
"background noise" viruses are commonly pres- 
ent in the animals used for the induction of 



142 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tumors, and of course in the subsequent passage 
materials as mentioned above. The extraneous 
viruses most commonly encountered in cancer sys- 
tems were the newer ones, such as polyoma, K 
virus, mouse reovirus and adenovirus; but this 
in part may be due to newly developed easily 
applied survey tools for these agents. Other vi- 
ruses encountered less often (perhaps because of 
comparatively less sensitive tools) were mouse 
hepatitis, mouse salivary gland virus, the newly 
discovered "thymic agent" (TA). Except in 
newborns, most of these viruses occur subclini- 
cally and latently. 

Medical Mycology 

Investigations on pathogenic fungi have in- 
cluded broad fields of research and although de- 
finitive goals have been reached in most of them, 
all will be continued in order to further exploit 
productive lines of investigation. In most cases 
new or additional species of pathogenic fungi 
will be used in investigations, or techniques will 
be altered to permit further development of ex- 
perimental studies. 

The antibiotic X-5079C was found to be fungi- 
static but not fungicidal and its apparent low 
degree of in vitro activity due to its decay in 
culture medium. The yeast form of Histoplasma 
capsulatum is much more sensitive to X-5079C 
than the mycelial form and an assay method, sen- 
sitive to 1 ug/ml using H. capsulatum, was de- 
veloped. X-5079C has low toxicity for HeLa cells 
and is active against H. capsuXatum grown in 
HeLa cells. 

A second strain of Coccidioides irrvmitis has 
been converted to serial culture in the spherule 
form. Quantitative measurements show the abil- 
ity of various carbon and nitrogen sources to 
support growth of spherule and mycelial forms 
of strain M-ll of C. irrvmitis. Only mannose is 
utilized as readily as glucose by spherules. Man- 
nose and fructose support growth of the mycelial 
form as well as does glucose. A substrate which 
preferentially supports growth of the spherule 
form was not found in this study. 

Spherules were utilized to immunize mice. An 
increased survivor rate in the immunized mice 
was noted after challenge with a lethal infecting 
dose and an earlier clearance of organs (negative 



cultures) in the immunized mice was observed 
after challenge with a sublethal dose. 

Cryptococcus Neoformans 

By titrating and plating out organs of experi- 
mentally infected mice, it was found that several 
minutes after Cryptococcus neoformans was in- 
jected either intravenously or intracerebrally into 
mice, the largest numbers of yeast cells had been 
retained in the lungs. The fungus population in 
the lung then decreases and 2-3 days after infec- 
tion multiplication in the brain is apparent. Al- 
though the interval from infection to death of 
infected mice varies with the strain of C. neo- 
formans, the numbers of yeast cells per gram of 
brain tissue are approximately the same regard- 
less of strain. 

Studies of the saprophytic occurrence in natural 
habitats of fungi which cause mycoses have con- 
tinued. Cryptococcus neoformans has been iso- 
lated from many additional collections of pigeon 
guano. When this material is collected from old 
pigeon nests and from roosting sites in hay mows 
of barns and upper floors of buildings, Histo- 
plasma has never been found. There is increasing 
circumstancial evidence that a presently unstudied 
pneumonic form of cryptococcosis has occurred 
in men heavily exposed to such material and that 
such epidemics have been erroneously diagnosed 
histoplasmosis. 

Emmonsia Crescens 

In collaboration with an investigator at the 
Rocky Mountain Laboratory a new species, Em- 
monsia crescens, was described. This fungus 
differs from the first species of Emmonsia 
(E. parva) in vivo and in vitro at 37° C. by its 
multinucleate condition (instead of uninucleate), 
its ability to produce the in vivo form in vitro 
at 37° O, and its greater size. E. parva conidia 
When inhaled or incubated at 37° C. increase in 
diameter from 2-4 u to 400-480 u. This 10 6 -fold 
increase in volume of a single cell is very unusual 
in the fungi. 

Staphylococcus Studies 

It has been established that staphylococcal 
penicillinase is associated with particulate mate? 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



143 



rial in the cell and thus an explanation has been 
given for the refractoriness in preparation of 
this enzyme by conventional methods. New and 
more potent inhibition of Staph, penicillinase 
have been uncovered and the hope remains and 
is heightened for the ultimate finding of a chemi- 
cally useful inhibitor. Further, sea water has 
been found to possess strong inhibitory activity 
against both penicillin-sensitive and penicillin- 
resistant staphylococci (phage type 80/81). 

Keal progress has been reported in the under- 
standing of iron metabolism in the staphylococci. 
As a direct result of continuing work dealing with 
mechanisms of the development of nonspecific 
immunity and in particular the function of the 
iron-transporting protein of plasma, siderophilin, 
fundamental observations on the effects of iron 
deficiency on the growth and metabolism of 
8. aureus have been reported. Work on the biol- 
ogy of the staphylococci so long neglected during 
the "antibiotic era" is cardinal to effective new 
therapy of staphylococcal infections. 

Streptococcal M Protein 

Progress has also been made on the search for 
better methods of isolation and purification of 
M protein of streptococci. These results are of 
obvious importance in the understanding of 



Group A streptococcal virulence. Further, highly 
interesting observations have been reported deal- 
ing with the mechanism and significance of the 
long-chain test for determination of anti-strepto- 
coccal immunity. 

Bacterial Metabolism 

Real understanding of the intimate mechanisms 
of energy metabolism in Hydrogenomonas in par- 
ticular and other bacteria and higher forms in 
general is closer as a result of work performed in 
this section this year. In an enormously compli- 
cated field, progress has occurred in the defini- 
tions of the essential reactions. 

Detoxification studies on potentially useful 
chemotherapeutic agents have continued and new 
and promising leads have been uncovered for 
agents active against bacteria, fungi, parasites, 
and, it should be added, against cancer as well. 
Several of the aforementioned detoxified com- 
pounds have passed preliminary screening proc- 
esses performed by the Cancer Chemotherapy 
Center. 

Pinpointing of the enzymic locus of discrimi- 
nation among hydrogen isotopes by pseudomonas 
has been reported this year. The area lies in 
formic acid metabolism. 



NATIONAL INSTITUTE OF ARTHRITIS AND 
METABOLIC DISEASES 



BASIC RESEARCH 

Introduction 

During the coming calendar year, NIAMD will 
attain its tenth birthday. The intramural struc- 
ture has completed a period of growth, which 
despite occasional growing pains, has been in the 
main satisfying to its personnel and productive 
of good science. Both the pains and the science 
have been reviewed in earlier issues of this annual 
report. 

We now look forward toward an era of limited 
physical growth. The laboratory and clinic space 
assigned to us is approximately saturated with 
men, with machines or with both. Indeed, in 
some areas the degree of crowding is such as to 
interfere with highest production. We can an- 
ticipate at this time but one increment in our 
space allotment, that portion of Building 2 cur- 
rently occupied by The National Institute for 
Dental Research. The intended disposition of 
this space will be considered subsequently. 

The fact that the limitation of physical space 
is now in sight and that a limitation in personnel 
must inevitably follow should not, of course, be 
taken to indicate a cessation of scientific and in- 
tellectual development. Indeed, we may expect 
that decline in architectural and personnel dis- 
tractions will direct an increasing fraction of our 
cumulative energy toward our prime business 
which is the conduct of research. 

Administration 

Over the past decade the administration of this 
Institute has been largely concerned with prob- 
lems of new recruitment, addition and fortifica- 
tion of sections and branches, acquisition and 
refurbishing of space, and purchase of the essen- 
tials of research equipment. Henceforward, at- 
tention will have to be directed chiefly toward 
replacement of lost personnel and of obsolescent 
equipment. 



It is our belief that in treating with excellent 
scientists, the best administration is the least 
administration. The mandate presented to the 
administrator is to tempt to his Institute research 
leaders of the very highest quality available, to 
supply them with the materials, support and en- 
vironment for optimal productivity, and then, to 
leave them alone. The only important control 
which administration may effectively exert over 
the choice of areas of research is in the selection 
of the disciplines in which senior recruitment is 
undertaken. Thereafter, one must have sufficient 
confidence in the scientist selected to place in his 
hands the choice of what experiments he shall 
perform. If he has been properly chosen, he will 
be best qualified to make this decision. The ad- 
ministrator of first-rate scientists has two impor- 
tant responsibilities; firstly, to resist the tempta- 
tion to meddle in the science of his staff, and 
secondly, to make no compromise with quality. 

In relation to the latter point, the accompany- 
ing opinion is expressed by the President's Sci- 
ence Advisory Committee: "In the advancement 
of science, the best is vastly more important than 
the next best. Mediocre research is generally 
worse than useless * * * It is therefore of first 
importance that national support * * * should 
aim at sustaining and reinforcing outstanding 
work wherever it may be f ound." 1 

Research and Education 

A recent report on scientific progress in the 
United States contains the following statement: 
"Yet in the long run it is dangerous to separate 
research in any field entirely from education. If 
a research field is to be attractive to good young 
men, it ordinarily needs roots in the universities. 
The pool of graduate students in our universities 
is the pool from which the scientists of the future 



i This quotation and the one which follows are from "Scien- 
tific Progress, The Universities and The Federal Government," 
Statement by The President's Science Advisory Committee, The 
White House, Washington, D.C., November 1960. 



145 



146 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



must come. These young people do not easily 
study what is not taught; they do not often learn 
the meaning of research which does not exist in 
their environment. A scientific field which has no 
research life in the universities is at a grave dis- 
advantage in recruiting new members. As learn- 
ing and teaching require research, so research, in 
the end, cannot be sustained without teaching. 
Hence it is always important for research instal- 
lations to maintain effective connections with 
students." 

It is the thesis of this report that basic research 
and graduate education go best together. This 
thesis is in harmony with what many of us be- 
lieve. It has been our hope to take steps, insofar 
as these are compatible with government regula- 
tions, to permit scientists in this Institute to reap 
the benefits normally reserved for members of 
university faculties. Among these the following 
may be listed : 

1. To facilitate access by the scientist staff to 
that most valuable and industrious population of 
junior investigators, the pre- and post-doctoral 
fellows. 

2. To permit to our scientists the continuous 
rejuvenation which comes from repeated exposure 
of an aging faculty to class after class of fresh 
and refreshing youngsters. 

3. To gratify, for those who need such gratifi- 
cation, the achievement of that "immortality" 
which derives from seeing one's teachings trans- 
mitted to succeeding generations of scientists. 

These and other related goals must be attained 
if NIAMD is to remain a most attractive en- 
vironment for our most productive scientists. To 
this end NIAMD has participated actively in all 
ventures in and about Bethesda relating to the 
teaching-learning function. The research Asso- 
ciate program is blossoming and in our Institute 
eight such Associates are presently housed, a new 
class of four being admitted each July. This 
program has attracted wide and favorable atten- 
tion in the best teaching hospitals and medical 
schools and the number of applicants each year 
is burgeoning. The courses sponsored by the 
Graduate School of the U.S. Department of 
Agriculture at NIH also continue to grow in en- 
rollment. NIAMD participates by supplying 
both students and teachers in this unusual edu- 
cational adventure. In addition, members of our 
scientist staff offer courses at local universities 



.and sponsor at Bethesda Ph.D. candidates en- 
rolled in these schools. All these arrangements, 
though less than ideal, serve to satisfy, to some 
extent, the goals mentioned above. Various cate- 
gories of summer students have also been accom- 
modated. 

Laboratory of Molecular Biology 

This is to be a new laboratory. Ultimately, 
five Sections are envisioned, tentatively to be 
named Metabolic Enzymes, Chemical Genetics, 
Physical Chemistry, Molecular Structure and 
Microbial Genetics. Two of these five sections 
will represent groups presently in other Labora- 
tories of our Institute who will move and expand 
in a new environment. Section Chiefs for two 
of the remaining three groups have been recruited 
and active steps are under way in the filling of 
the remaining vacancy. 

The guiding philosophy in the staffing of this 
new laboratory has been to collect a group of 
outstanding, energetic and enthusiastic investi- 
gators with common goals but diverse technical 
backgrounds. All the groups will have interests 
in and about the biological role of macromolecules 
such as nucleic acids and proteins. They will all 
be concerned, more or less, with the biogenesis, 
structure and function of these molecular species. 
They will bring to this interest the skills and 
ideas of the crystallographer, the spectroscopist, 
the enzymologist, the geneticist and others. It is 
hoped and expected that the interchange of ideas 
among these groups of investigators will result 
in a whole which is far greater than the algebraic 
sum of its parts. 

Organisational Matters 

The retirement of Dr. Kalph Lillie required a 
review of the Laboratory of Pathology and His- 
tochemistry which he had headed for many years. 
Dr. L. L. Ashburn was persuaded to become the 
new Chief of the Laboratory, assisted by Dr. 
Gert Laqueur. To accommodate the needs of the 
several scientists, sections were rearranged and 
Section Chiefs were designated as follows: Dr. 
Spicer, Biophysical Histology; Dr. Highman, 
Pathologic Anatomy ; Dr. Brecher, Hematology ; 
Dr. Glenner, Histochemistry ; Dr. Sokoloff, Rheu- 
matic Diseases. 

The retirement of Dr. Nathan Eddy deprived 
the Section on Analgesics of the Laboratory of 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



147 



Chemistry of its chief. Happily, Dr. Everett 
May, long associated with Dr. Eddy, will serve 
as chief of the section, renamed Section on Medi- 
cinal Chemistry. 

The Pediatric Metabolism Branch under Dr. 
Paul di Sant'Agnese is now well launched. This 
clinical branch has found common research inter- 
ests with several of our basic science laboratories 
and its members have entered upon a number of 
interesting collaborative ventures. 

A number of distinguished Visiting Scientists 
have come to NIAMD during the past year. 
Dr. E. G. L. Bywaters of London spent 3 months 
working with members of the Arthritis and 
Rheumatism Branch. Dr. Ephraim Racker of 
New York visited for several months with mem- 
bers of the Laboratory of Biochemistry and Me- 
tabolism, where the influence of his vast knowl- 
edge and fine critical judgment was greatly 
appreciated. In the same laboratory, Dr. Ernst 
Simon of Rehovot, Israel, has spent a year in 
many active collaborations with various members 
of NIAMD in and about his own research inter- 
ests. Dr. Hin Tjio of Saragossa, Spain has been 
working in the Laboratory of Pathology and 
Histochemistry. His unique contributions in the 
area of chromosome anatomy have brought to 
him many consultations and proposed collabora- 
tions. 

Intramural scientists have also participated in 
a variety of extracurricular functions. Among 
these may be mentioned Dr. Bunim's delivery of 
the Heberden Oration and Dr. Heppel's presen- 
tation of the 10th N.I.H. Lecture. 



Laboratory of Nutrition and Endocrinology 

The primary emphasis of the work in the labo- 
ratory is directed toward the elucidation of the 
mode of action of nutrients and hormones. To 
this end intact animals are intensively studied 
to determine the effect that dietary or environ- 
mental factors may have on the animals' physio- 
logical economy. At the termination of these 
studies, a variety of tissues are examined bio- 
chemically or histologically to secure a more 
exact description of the role played by nutrients 
and hormones. It is anticipated that these studies 
will also provide clues and suggest additional 
investigations for those scientists who are inter- 



ested in the more restricted areas of biochemistry 
or physiology. 

During the past year, the laboratory has in- 
creased its efforts in the field of biochemistry. 
Because of the large amount of biochemical work 
being done on each animal, there has been a 
reduction in the space devoted to the care of the 
animals and an accompanying increase in labo- 
ratory area. 

Nutrition 

Vitamin E and Factor 3. Shortly after vita- 
min E was first reported as an essential nutrient 
for normal reproduction of rats, this vitamin be- 
came the center of a controversy. A number of 
investigators claimed that some animal species 
did not require the vitamin at all. Later, other 
workers showed that the functions ascribed to 
vitamin E could be assumed by a variety of com- 
pounds all of which were effective antioxidants. 
Although somewhat abated, the controversy still 
continues with the arena reduced from the intact 
animal to individual tissues or cellular compo- 
nents. 

A few years ago, Dr. John G. Bieri showed 
that chicks could be raised to adulthood and 
could produce fertile eggs when fed a diet devoid 
of any detectable vitamin E. After a year on 
the deficient diet, the tissues of the birds con- 
tained no vitamin E as shown by analysis. In 
continuing this work, Dr. Bieri developed an in 
vitro test system using the auto-oxidation of liver 
cell mitochondria as an index of tissue vitamin E 
deficiency. The lipids in mitochondria from 
vitamin E-depleted animals readily auto-oxidize 
whereas mitochondrial lipids from animals fed 
an ordinary diet containing vitamin E do not. 
If the diet is devoid of unsaturated fats and vita- 
min E there is a substantial reduction in peroxi- 
dation. 

The addition of vitamin E or a number of anti- 
oxidants directly to the mitochondrial system 
reduces the peroxidation seen in the in vitro test. 
The addition of selenium or cystine to the in 
vitro system prepared from chick livers has no 
effect but when these compounds are fed to the 
animals, their liver, heart, and kidneys showed 
antioxidant activity. These observations suggest 
that when selenium or cystine are fed, the chick 



148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



forms a substance which functions to reduce the 
level of peroxidation. 

The chick has a very limited capacity to store 
the biologically active form of selenium in its 
body. This was shown by Dr. Bieri when he fed 
three groups of day-old chicks different levels of 
selenium in a vitamin E-free diet for one week. 
At the end of this period, the selenium was re- 
moved from the diet. All groups developed 
symptoms of a combined vitamin E and selenium 
deficiency at approximately the same time regard- 
less of the level of selenium present in the diet 
during the one week feeding period. 

Dr. Bieri has concluded from his own work 
and that of other scientists that the primary func- 
tion of vitamin E in the chick is that of a tissue 
antioxidant. This vitamin is important in this 
respect not only in reducing the oxidation of 
lipids in the diet and gastrointestinal tract but 
in the tissues of the animal as well. 

In the course of the above work an analytical 
method was developed for chromatographically 
separating «-tocopherol from the unsaponifiable 
fraction of animal tissues. By this means as little 
as 2 /»g. of a-tocopherol can be detected. The 
procedure can be applied to 0.4 gm. of tissue. 
Ubiquinone (or coenzyme Q) can also be deter- 
mined by this method. 

In January 1957, Drs. Klaus Schwarz and Cal- 
vin M. Foltz showed that dietary liver necrosis 
in rats could be prevented or cured by adding 
very small amounts of certain inorganic selenium 
compounds to the diet. The concentrates of Fac- 
tor 3 prepared from natural products contained 
selenium in an organic form. Smaller amounts 
of Factor 3 than of inorganic selenium were re- 
quired in the diet to prevent dietary liver necro- 
sis. A variety of organo-selenium compounds 
synthesized by Prof. Arna Fredga of the Uni- 
versity of Upsala and assayed by Dr. Schwarz 
indicated that optimal Factor 3 potency occurred 
in di-seleno, dibasic acids containing 4 or 5 car- 
bon atoms. The straight chain compounds were 
more active than branched chains. The di-seleno 
acids are converted in the animal to seleninic 
acids before they become biologically effective. 
The most active compound is benzyl-A-mono- 
selenovaleric acid which is close to the activity 
of Factor 3. 

In an attempt to determine how selenium func- 
tions, Dr. Fiorenze Stirpe (visiting scientist) 



studied the incorporation of isotopically labelled 
valine into the tissues of rats maintained on the 
necrosis-producing ration (Torula yeast ration). 
This work indicated that the rate of incorpora- 
tion of the valine into protein was enhanced in 
the animals with necrosis but it was not affected 
by dietary supplements of liver protective sub- 
stances such as Factor 3 or vitamin E. The in- 
crease of amino acid uptake was related to in- 
creased rate of protein turnover which appears 
within a few days after the rats are put on the 
Torula yeast diet. 

Work by Dr. Trygve Tuve with fresh Baker's 
yeast showed that the cells incorporate trace 
amounts of radioselenite when incubated in the 
presence of glucose. After a short lag period, 
the cells incorporate the selenium at a steady 
rate which depends upon the level of selenite in 
the medium. Most of the radioactive selenium 
(92 to 94 percent) appeared to be in a "protein- 
bound" fraction (precipitable with trichloroacetic 
acid) . Chromatographic analysis of hydrolysates 
of this fraction showed an organoselenium com- 
pound that differs from the selenium compounds 
previously found in proteins. Fractionation of 
the trichloroacetic acid soluble material of the 
cells indicated the presence of another new or- 
ganoselenium compound. The selenium was not 
present as an analogue of cystine, methionine, 
gluthathione, or selenium, di-, or tetra-cysteine. 
The two unknown compounds were also detected 
in rat urine following an injection of radio- 
selenium. These substances were also present in 
the trichloroacetic acid extracts of liver and kid- 
ney homogenates. 

There are a number of areas in the world in- 
cluding the Pacific Northwest where lambs and 
sheep develop white muscle disease resulting in 
large economic losses to the farmers. The addi- 
tion of small amounts of selenium to the rations 
fed the pregnant dams in these areas eliminated 
the difficulty. Pasture grasses from the North- 
west were shown by Drs. Schwarz and John E. 
Schubert (guest worker) to contain adequate 
amounts of selenium, but it was not always avail- 
able to the animal. 

In previous studies, vitamin E-deficient rat 
liver homogenates showed a marked decline in 
the rate of oxidation of a-ketoglutarate and suc- 
cinate. Work during the past year by Dr. Lau- 
rence M. Corwin showed that the same is true for 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



149 



fumarate, malate, pyruvate with a catalytic 
amount of malate, a-hydroxybutyrate, and gluta- 
mate. Vitamin E when added either to the diet 
or to the Warburg flask completely prevented the 
decline in the rate of oxidation of most of the 
above systems. Compounds such as glutathione, 
BAL (British antilewisite), menadione, N,N'- 
diphenyl-p-phenylenediamine, and methylene 
blue which maintained the integrity of free sulf- 
hydryl groups were also effective in overcoming 
the decline in oxidation. The effects are not re- 
lated to auto-oxidation, since the metabolic im- 
pairment can be prevented by levels of the pro- 
tective compounds which are not effective in 
preventing peroxide formation. Additional work 
suggested that tocopherol shields the vicinal 
dithiol groups in the dehydrogenases and other 
sulfhydryl groups essential for the enzyme ac- 
tivity and thus spares them from inactivation by 
inhibitors such as cadmium and arsenite. Since 
microsomes are required for decline to occur, the 
nature of the microsomal agent actively precipi- 
tating the respiratory decline was studied. The 
agent in the microsomes is not stable to boiling 
nor can it be removed by EDTA (ethylenedi- 
aminetetraacetic acid) and subsequent dialysis. 

Folic Acid. During the past year significant 
progress has been made in the isolation of the 
folic acid derivative that occurs naturally in 
liver. A major breakthrough in this problem 
occurred when it was demonstrated by Drs. John 
Iveresztesy and Kenneth O. Donaldson that tetra- 
hydrofolic acid could be enzymatically converted 
to the prefolic acid form as evidencer by chroma- 
tographic and microbiological assay (performed 
under the direction of Mr. Howard A. Baker- 
man). The enzymatic system requires a reduced 
diphosphopyridine nucleotide-menadione reduc- 
tase system. These studies indicate that this pre- 
folic compound is a reduced tetrahydrofolic acid 
derivative. An attempt is being made to increase 
the scale of the enzymatic reaction so that suffi- 
cient amounts of the reduced tetrahydrofolic acid 
can be isolated and identified. 

Although the folic acid activity of many cells 
and tissues is known, very little information is 
available about the different folic acid compounds 
which are present in the cells and tissues. Dr. 
Milton Silverman has developed procedures for 
the quantitative identification of the known 



folic acid-like compounds. With these techniques, 
Dr. Silverman has shown that in some leukemic 
cell lines (with Dr. Lloyd Law, NCI) the major 
folic acid form is N 10 -formyl tetrahydrofolic acid 
(80 percent). The other components consist of 
small amounts of N 5 -formyltetrahydrofolic acid 
and unsubstituted tetrahydrofolic acid. In con- 
trast to the situation in the tumor cells, the liver 
of the host mouse contains primarily the prefolic 
acid compound being studied by Drs. Iveresztesy 
and Donaldson with small amounts of the tetra- 
hydrofolic acid compounds mentioned above. 
These results suggest that the functional form 
of folic acid in the tumor cells is a monogluta- 
mate derivative reduced to the tetrahydro level. 
This is in contrast to a generally accepted propo- 
sition that the physiologically active forms of 
folic acid exist as polyglutamates. Similar stud- 
ies are underway on several bacterial species. 
This work (with Dr. Bernard T. Kaufman and 
Mr. Bakerman) has shown that these cells not 
only contain most of the known folic acid com- 
pounds but also several derivatives which have 
not been completely characterized. 

In the presence of reduced triphosphopyridine 
nucleotide, folic acid is reduced to tetrahydrofolic 
acid by a protein fraction from chicken liver. 
N 10 -f ormylfolic acid is also reduced to the tetra- 
hydro level by chicken liver extracts. However, 
the mechanisms involved in these two reductions 
appear to be different. Although the requirement 
for TPNH (triphosphopyridine nucleotide) is 
common to both systems, the reduction of the 
formylated derivative is stimulated by the pres- 
ence of free folic acid and citrate or versene. 
The enzymatic components involved in the reduc- 
tion of N 10 -formylfolic acid are being purified by 
Drs. Silverman and Kaufman. 

A number of folic acid antagonists have been 
used in the treatment of a variety of diseases 
and in studies directed toward the elucidation 
of the metabolic function of folic acid. Dr. Roy 
Kisliuk (Visiting Scientist) succeeded in reduc- 
ing some of the folic acid antagonists (aminop- 
terin and amethopterin) to their tetrahydro de- 
rivatives. These compounds appear more closely 
related to the active form of the vitamin than 
the previously used antagonists. The reduced 
antagonists proved more effective in overcoming 
the activity of folic acid than the unreduced forms 
both in bacterial cells and in chicks (Dr. M. R. 



150 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Spivey Fox). These results with the observation 
of other investigators (that aminopterin and 
amethopterin can be reduced by cells) suggest 
that in vivo these inhibitors exert their effects 
after reduction to the tetrahydro form. 

Vitamin B 12 . Previous work by Dr. Fox showed 
that a severe deficiency of vitamin B i2 could be 
produced in the chick by feeding it a diet high 
in fat, low in methionine, and free of the vitamin. 
The deficient chicks exhibited poor growth and 
an impaired metabolism of histidine as shown by 
a high excretion of formiminoglutamic acid. 
Dietary supplements of either vitamin B i2 or 
methionine overcame both signs of deficiency. 
The intact methionine molecule is required for 
these effects since homocystine, cystine, choline, 
or betaine were inactive. Thymidine, which can 
replace vitamin B 12 under some conditions, had 
no effect on the excretion of formiminoglutamic 
acid. The high fat level in the deficient diet was 
necessary for poor growth but not for the excre- 
tion of formiminoglutamic acid. 

Germ-Free Studies. The germ-free animal pre- 
sents a unique opportunity to secure unequivocal 
answers to a variety of questions relating to the 
possible contribution of the intestinal flora to 
the physiological economy of the animal. In ad- 
dition thereto, this opportunity has resulted in 
problems which are uniquely related to germ-free 
animals. One of these relates to the difficulty in 
meeting completely the germ-free animals' nutri- 
tional requirements. Work by Dr. Floyd S. Daft, 
Mr. Ernest G. McDaniel, and Mr. James C. Smith, 
Jr., has resulted in the development of diets 
which produce reasonably good growth in the 
guinea pig. These animals, however, develop 
caeca which in some cases become so large that 
they represent 43 percent of the total body weight 
(in a 21-month-old-male). In conventional ani- 
mals, the weight of the caecum is usually 2 to 3 
percent of the body weight. Since there is a 
possibility that autoclaving the diet may destroy 
certain essential and as yet unreocgnized nutri- 
ents and that these nutrients may be required in 
maintaining the caecum at normal size, a number 
of germ-free guinea pigs were fed supplements 
of raw fresh eggs, whole human blood, raw tis- 
sue, and feces from germ-free rats. These natu- 
ral products were tried since they were sterile 



originally and for this reason they could be 
brought into the germ-free tank without being 
autoclaved. None of these substances was effec- 
tive in reducing the size of the caecum. There 
was actually an indication that the guinea pigs 
consuming the rat feces showed an aggravation 
in the enlargement of the caecum. There is a 
possibility that the rat feces fed the guinea pigs 
may have increased the amount of trypsin in the 
lower part of their intestinal tract. This is based 
on the observation of Dr. Bengt Gustaffson (vis- 
iting scientist) that the feces from germ-free rats 
contain large amounts of trypsin whereas the 
feces from conventional animals contain none of 
this enzyme. 

The enlarged caecum is not the result of the 
germ-free state since a number of conventional 
guinea pigs that were removed from their mothers 
by caesarian section and fed nothing but auto- 
claved diets showed similar enlargement of their 
caeca. The removal of the major portion of the 
caecum from day-old germ-free guinea pigs at 
first appeared to keep the caeca small but until 
the animals are sacrificed no conclusive answer 
will be available. An interesting incidental ob- 
servation of the latter study was the high mor- 
tality (80 percent) among the conventional 
guinea pigs on whom the caecaectomy was per- 
formed compared to the low mortality (20 per- 
cent) among the germ-free animals. The conven- 
tional animals died within less than 24 hours of 
the operation showing severe shock so it is doubt- 
ful whether infection can explain the differential 
mortality. 

Protein. Dr. J. N. Williams, Jr. is studying the 
effect of a prolonged dietary protein deficiency 
on the changes in the composition of the liver of 
rats. Within 10 days after the animals were put 
on the protein-free ration, they began to show 
changes, some of which progressed throughout 
the 72 days of the experiment. The number of 
cells in the liver was estimated from the concen- 
tration of deoxyribonucleic acid. On this basis, 
the number of cell per unit weight of liver 
almost doubled after 72 days of protein deficiency. 
This change was primarily a reflection of inani- 
tion and not of protein deficiency per se since 
the same change was shown by the animals fed 
a complete diet in amounts equal to that con- 
sumed by the protein-deficient rats. The nitro- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



151 



gen content of the individual liver cell in the 

< protein-deficient group was reduced to 60 percent 
of that in the rats fed a complete diet on an 

' ad libitum, basis; the comparable value for the 
pair-fed controls was 85 percent. The total num- 
ber of liver cells in the protein-deficient and 

: pair-fed control animals showed a progressive 
reduction throughout the 72-day period to 70 
percent of the ad libitum controls. The ratio of 

1 liver weight to body weight increased markedly 
in the protein-deficient rats while little change 

' occurred in the pair-fed controls. 

' The deprivation of dietary protein should in- 
fluence the animals' ability to synthesize the en- 
zymes involved in energy transformations. To 

; evaluate this proposition, Dr. Williams deter- 
mined the activity of the individual components 

' of the succinic oxidase system in his protein- 
deficient rats. The activity of succinic oxidase 
and succinic dehydrogenase per liver cell de- 
creased to one-half of normal by the 10th day of 
the protein deficiency. Thereafter, the cells ap- 
peared to resynthesize these enzymes so that by 
the end of the experiment their activities were 
about 75 percent of normal. Throughout the 
study, there was no change in the antimycin 
A-sensitive activity (a measure of the flow of 
electrons between cytochromes-& and <?i) or of 
cytochrome-c concentration within each cell. 
About 85 percent of the cytochrome oxidase 
activity was lost from the cell after 46 days on 
the protein-deficient diet. In spite of the large 
reduction in the cytochrome oxidase activity, 
there was still sufficient enzyme to maintain suc- 
cinic oxidase activity at a high level. These 
studies indicate that each individual enzyme of 
the same mitochondrial system responds differ- 
ently to a protein deficiency. 

Dr. Peter G. Condliffe, during the tenure of a 
fellowship sponsored by the National Foundation, 
spent one year at the Carlsberg Laboratory in 
Copenhagen. While there he studied the ex- 
changeable hydrogen atoms in orosomucoid which 
is a glycoprotein present in human plasma to the 
extent of 0.05 to 0.1 percent. It is the principal 
protein excreted in the urine of patients with 
nephrosis. Dr. Condliffe found that at neutrality 
and 37° all available hydrogens exchanged within 
two hours but a number, approximately equal to 
the number in the peptide moiety, do not ex- 
change instantaneously. The latter hydrogens 



probably represent bonds involved in the sec- 
ondary structure of the molecule. An helical 
structure would explain this observation. Con- 
firmation of this hypothesis will be sought in 
studies of the optical rotatory dispersion of oroso- 
mucoid. During the course of this work a new 
method was developed for the preparation of the 
protein which involves the use of an alcoholic 
fraction of plasma protein- VI (Cohn's fraction). 
After the alcohol is removed and the protein 
dissolved in water, the solution is dialyzed against 
a phosphate buffer. The equilibrated solution is 
put through a diethylaminoethyl cellulose column 
which adsorbs the orosomucoid. Elution is ac- 
complished with a sodium phosphate solution. 

Lipids. Drs. Robert O. Scow, Sidney S. Cher- 
nick, and Martin Rodbell (NHI) found that 
when an emulsion of cottonseed oil labelled with 
C 14 -tripalmitin was perfused through the liver 
of a rat, the disappearance of the labelled tri- 
palmitin was proportional to its concentration in 
the perfusate. This was true for concentrations 
up to 7.4 milliequivalents of ester per liter. 
Above this level, the liver removed the lipid at a 
constant rate of 10 microequivalents per gm. of 
liver per hour. The labelled ester removed by 
the liver was recovered primarily as neutral and 
phospho lipids (60 to 90 percent) and to a lesser 
extent as ketone bodies (11 percent) and carbon 
dioxide (2 percent). Although both the Kupfer 
and parenchymal cells removed the triglyceride 
without de-esterification and metabolized it, the 
parenchymal cells accounted for 75 to 80 percent 
of the activity. 

Plasmalogens comprise a group of phospho- 
lipids which give rise to a long chain aldehyde, 
a fatty acid, glycerol, a base, and phosphoric 
acid on acid hydrolysis. Previous work by Drs. 
Carl E. Anderson (Visiting Scientist) and Wil- 
liams indicated that liver contains an enzyme 
which splits the vinyl ether linkage by which the 
aldehyde is attached to the plasmalogen. To fa- 
cilitate this work, a method was devised for the 
separation of the plasmalogens from other alde- 
hydrogenic compounds in the lipid extract. Sili- 
cic acid chromatography under special conditions 
proved satisfactory for this procedure. With this 
technique in hand, it is now possible to proceed 
with the enzymatic study. 



152 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Glucose Tolerance Factor. Previous work by 
Drs. Walter Mertz and Schwarz showed that 
when rats were raised on certain diets, their rate 
of removal of intravenously injected glucose was 
reduced from a control value of 4 to 4.5 percent 
to 2.8 percent per minute. They found that 
when trivalent chromium was added to the diet, 
the removal rate of the glucose was restored to 
the control value. The epididymal fat pads re- 
moved from rats showing the reduced rate of 
glucose removal utilized less glucose than was 
true of the pads from rats fed the control diets. 
The glucose uptake could be increased to normal 
by the addition of chromium to the excised tis- 
sue. The activity of a number of different chro- 
mium (III) complexes in the tissue assays was 
almost identical to the activity seen in the in vivo 
experiments. These findings indicate that differ- 
ences in the glucose tolerance factor (GTF) ac- 
tivity of the chromium complexes in the in vivo 
studies cannot be explained solely on differences 
in intestinal absorption. 

The work of Dr. Mertz indicated that insulin 
was required in the in vitro system to show the 
stimulatory effect of chromium on the incorpora- 
tion of labelled glucose and to a lesser extent of 
acetate into the lipids of adipose tissue. Labelled 
galactose was used to determine whether chro- 
mium had any effect on the transport of nutrients 
across the cell membrane. In such studies the 
adipose tissue from rats raised on the GTF-de- 
ficient diet showed a reduced rate of galactose 
entry. The addition of 0.01 ng. of chromium in- 
creased tissue galactose by 56 percent after one 
hour of incubation. At that time the tissue 
water in the controls was 67 percent saturated 
with galactose whereas it was 100 percent satu- 
rated in the chromium-supplemented tissues. 
These studies suggest that chromium may func- 
tion close to the site of action of insulin, i.e., in 
increasing the entry of sugars into cells. 

Obesity. The obesity produced in a variety of 
strains of rats by feeding a high-fat diet has a 
genetic component to it as shown by the work of 
Dr. Richard S. Yamamoto. He found that a 
hybrid produced by crossing the Sprague-Dawley 
with the NTH black rat did not become obese 
when fed the high-fat diet even though the 
progenitors of the strain did. The level of fat 
in the diet has no effect on body composition, 



plasma lipids or liver composition of these hy- 
brid rats. An extension of these studies showed 
that an obese strain of mice (STR/1N) had 
higher levels of lipids in their plasma than mice 
of the lean strains (DBA/2JN and A/LN). The 
difference in the lipid levels was primarily due 
to neutral lipids. Genetic studies (with Dr. Leon 
Sokoloff ) showed that the obesity and high plas- 
ma lipid levels were dominant traits. These 
studies also showed that the high hemoglobin 
levels and hematocrit in the A/LN mice are domi- 
nant traits as shown by crossing these mice with 
the STR/lN mice which have low blood levels. 
In all groups, the hemoglobin level was directly 
related to the hematocrit. 

Guinea Pigs. The guinea pig has a higher re- 
quirement for dietary protein than most other 
laboratory animals as shown by the work of Dr. 
Mary E. Reid (Guest Worker). Maximum rates 
of growth were secured when the diet contained 
35 percent of protein either as casein or as a puri- 
fied soy protein. Good growth was also secured 
when the diet contained 20 percent protein, pro- 
vided the casein was supplemented with 1 percent 
L-arginine or the soy protein with 0.5 percent 
DL-methionine. These studies suggest that, as 
far as growth is concerned, the guinea pig has a 
high requirement for certain amino acids and 
not for protein per se. Evidence is also being 
accumulated by Dr. Reid that as the level of 
dietary protein is increased, the ratio of the 
weights of the liver, testes, and spleen to body 
weight increases. 

Rabbits. Previous work by Dr. Olaf Mickelsen 
and Miss Jeanne M. Reid indicated that although 
rabbits did not manifest the typical deficiency 
symptoms when fed a thiamine-free diet for long 
periods, they did show a reduction in urinary and 
fecal excretion and reduced levels of the vitamin 
in the brain. In an extension of this work, young 
rabbits were fed a thiamine-free diet supple- 
mented with the vitamin antagonist pyrithiamine. 
Six of the 7 rabbits developed typical neuro- 
logical symptoms beginning on the 18th day. 
Four of these rabbits died within 24 hours after 
first exhibiting the symptoms; the two remain- 
ing animals showed dramatic responses to thi- 
amine injected intraperitoneally. Pyrithiamine 
increased the urinary excretion of thiamine, of- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



153 



fering additional support for the hypothesis that 
this antagonist releases the vitamin from body 
cells. 

Endocrinology 

Experimental Diabetes. One of the major prob- 
lems still confronting the clinician who treats 
large numbers of diabetics is the sudden and ap- 
parently uncontrollable ketosis which develops 
in some patients. Considerable effort has been 
devoted by Drs. Scow and Chernick to the eluci- 
dation of the physiological factors in the dia- 
betic rat which produce severe ketosis. Earlier 
work by this group showed that when insulin 
was withheld for 17 hours or more from the hy- 
pophysectomized-pancreatectomized rat, severe 
ketosis developed following the administration 
of glucocorticoids. Growth hormone had no ef- 
fect in these animals. However, when these dia- 
betic rats were treated with insulin, and shortly 
thereafter injected, then both growth hormone 
and glucocorticoids were necessary to produce ke- 
tosis. Both hormones were also needed to pro- 
duce severe ketosis in the fasting pregnant rat. 
In both cases, insulin quickly suppressed the ke- 
tosis. These findings suggest that growth hor- 
mone acts as a ketogenic agent by masking the 
action of the traces of tissue insulin but this 
occurs only when the level of insulin is low. 

The above findings have been confirmed in the 
rat made diabetic by phloridzin. In this animal 
either insulin or bilateral nephrectomy (to pre- 
vent the loss of sugar) prevented the develop- 
ment of ketosis. The phloridzin-diabetic rat also 
required both glucocorticoids and growth hor- 
mone before severe ketosis developed. This ani- 
mal differed from the pancreatectomized rat in 
that there was no accumulation of lipids in the 
blood and liver when the blood ketone levels ex- 
ceeded 40 mg. percent. 

The work of Drs. Scow, Chernick, and Ernst 
Simon (visiting scientist) suggests that the dia- 
betogenic effect of mannoheptulose is due to its 
suppression of insulin release from the pancreas. 
When 400 mg. of mannoheptulose, a seven carbon 
sugar isolated from avocados, were injected into 
a rat, it immediately developed increased levels 
of blood glucose and ketone bodies. There was 
no change in the blood lipids. The effect of the 



mannoheptulose waned within 2 hours at which 
time its blood concentration fell below 40 mg. 
percent. Mannoheptulose is an isomer of sedo- 
heptulose, an important intermediate in the pen- 
tose shunt. Sedoheptulose differed from manno- 
heptulose in not raising the blood sugar level. 
Sedoheptulose resembled glucose in lowering the 
blood ketone level. When fasting rats were given 
glucose by stomach tube, the blood glucose level 
was increased and the blood ketone level de- 
creased. When mannoheptulose was given with 
the glucose, the blood glucose level was greatly 
augmented but the blood ketone level remained 
elevated until the mannoheptulose had practically 
disappeared from the blood. Insulin injected in- 
to the fasting rats lowered both the blood glucose 
and ketone levels even when mannoheptulose was 
given. These findings indicate that exogenous 
insulin can overcome the action of mannoheptu- 
lose and that the latter has no effect on the ac- 
tion of insulin once it is released from the pan- 
creas. Additional studies indicated that manno- 
heptulose had no effect on the blood levels of 
both glucose and ketone bodies in the pancreatec- 
tomized-diabetic rat. 

Dr. Scow is developing a technique for infus- 
ing the pancreas in situ. In the rat, this in- 
volved removal of the spleen and all the pancreas 
except that which receives its blood supply from 
the splenic artery. A polyethylene tube was in- 
serted into a small branch of this artery near 
its origin. With this setup it soon became evi- 
dent that the solutions injected into the artery 
did not mix completely with the blood, conse- 
quently only a small portion of the remaining 
pancreas was being infused. When this difficulty 
is overcome, the preparation will be used to 
study the effect of mannoheptulose infusion on 
insulin release from the pancreas. 

Dr. Scow and Andre Kobert (Guest Worker) 
have developed a technique for perfusing an iso- 
lated segment of adipose tissue. A long finger of 
fat attached to the mesometrium of female rats 
proved most satisfactory for this purpose. Sev- 
eral problems have developed such as hemolysis, 
blood clotting in the apparatus and edema of the 
perfused tissue. All but the last have been over- 
come. 

The work of Drs. Evelyn Anderson and Robert 
W. Bates during the past year suggests that the 
action of orinase on the release of insulin is still 



154 



ANNUAL REVIEW OF INTRAMUEAL RESEARCH 



unsettled. In some experiments with dogs, ori- 
nase has increased the level of insulin in the 
peripheral blood while in others, orinase has had 
no effect. One of the major problems in this 
study is the method of extracting the insulin 
from the blood prior to bio-assay. A number of 
extracts have killed the test animals and for this 
reason the extraction procedure is currently un- 
der intensive study. 

Pituitary Hormones. For the past 7 years Drs. 
Kobert W. Bates and Peter G. Condliffe have pre- 
pared their thyroid stimulating hormone (TSH) 
concentrates from two lines of pituitary tumors in 
mice. These tumors now show a decreasing con- 
centration of TSH. Attempts have been made 
over the past 2 years to locate new tumor lines 
with a high concentration of TSH. Of some 
30 primary pituitary tumors that have been trans- 
planted, only 4 or 5 have high concentrations of 
TSH. Mice with the latter tumor lines have 
enlarged bile ducts and elevated blood levels of 
TSH. 

A disappointing observation in this work is 
the instability of the dry TSH concentrates pre- 
pared from bovine pituitary. These concentrates 
regardless of whether they were stored at room 
temperature or in a vacuum dessicator at 1°C. 
all showed a 50 percent reduction in their ac- 
tivity. It is not known as yet whether or not 
the instability of these preparations is species- 
related as is the case for the prolactins. Prolac- 
tin preparations from sheep pituitaries are more 
stable than those from bovine glands, especially 
during fractionation in acid ethanol or acetone. 

The plasma of rats having large transplantable 
"mammotropic" pituitary tumors has been shown 
by Dr. Bates to contain as much prolactin on a 
dry weight basis as does the tumor powder. 
This is the first clear evidence that mammary 
development in rats with tumors is associated 
with increased prolactin levels in the blood. 

Dr. Bates has shown that the injection of 
thyroxine and prednisone or these combined with 
either growth hormone or prolactin had only a 
small effect on body weight gains and food in- 
take of hypophysectomized pigeons. When all 
four substances were given, 20 jug. of the mixture 
produced a greater effect than 1 mg. of either 
growth hormone or prolactin alone. The relative 
increases in weight of the gastrointestinal tract, 



liver, pancreas, and kidney were similar to but 
greater than the increase in body weight which 
confirms the splanchnomegalic effect of pituitary 
hormones in pigeons. 

Even when the pars distalis is removed from 
pigeons, the injection of such varied substances 
as insulin and formaldehyde will still produce 
a hypertrophy of the adrenals. The work of 
Dr. Richard A. Miller (visiting scientist) showed 
that when the infundibular process was removed 
with the pars distalis, thereby removing the 
source of ACTH, the weight of the adrenals 
doubled spontaneously in a certain percentage of 
the pigeons and in all birds if formaldehyde was 
injected. 

Evidence has been secured by Drs. Evelyn 
Anderson and Richard C. Grindeland (guest 
worker) that rats with mammotropic pituitary 
tumors continue to grow after hypophysectomy 
and that the rate of corticosterone production 
continues at a high rate. These observations sug- 
gest that these pituitary tumors elaborate growth 
and adrenocorticotropic hormones. 

Steroids. The work of Dr. Hildegard N. Wilson 
shows that patients with adrenocortical carci- 
noma have (1) a 3- to 10-fold increase in the 
total amount of A 5 -pregnenolone available for 
steroid hormone synthesis; (2) an increased pro- 
portion of the total synthesis going into the "an- 
drogen" pathway, especially in a virilized pa- 
tient; (3) a large increase in dehydroepiandro- 
sterone which could not be transformed further 
and was excreted as such; (4) an increase in the 
proportion of 11-deoxycortisol which was not 
hydroxylated on carbon 11 to form Cortisol; (5) 
an increase in the proportion of progesterone not 
converted to Cortisol. These studies suggest that 
certain enzymes may not be functioning properly 
in adrenal carcinoma tissue thus preventing the 
synthesis of certain corticosteroids in amounts 
equal to that produced by normal adrenal tissue. 

The urine of three patients with Klinefelter's 
syndrome studied by Dr. Wilson showed a reduc- 
tion in androgens and Cortisol metabolites. These 
patients with hypogonadism, gynecomastia and 
high titers of gonadotropins presumably have an 
enzymatic defect which affects the early stages 
of steroid synthesis either in the adrenals or 
gonads. 

It was shown by Drs. Anderson and James 



NATIONAL, INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



155 



Davis (NHI) that aldosterone secretion is not 
controlled by a humoral substance elaborated in 
the midbrain as postulated by other workers. 
Dogs with lesions in the midbrain secrete from 
the adrenal gland normal amounts of aldosterone. 
The rate is increased following hemorrhage. 

A micro method for the assay of ACTH has 
been developed by Miss Frances E. Wherry and 
Drs. Anderson and Bates. This method is 10 
times more sensitive than presently available 
methods. It is based on the increase in corti- 
costerone in adrenal venous blood of the hypo- 
physectomized rat following an intravenous in- 
jection of ACTH. Methods for the extraction of 
ACTH from plasma are being developed so that 
the levels of this hormone in plasma can be de- 
termined. 

Laboratory of Biochemistry and Metabolism 

Carbohydrate Metabolism: Reactions involving 
carbohydrate polymers. 

Glycogen isolated from animal tissues follow- 
ing extraction with cold acid has been found to be 
of appreciably larger average molecular size 
than that isolated by extraction with hot concen- 
trated alkali. Isolation and identification of the 
products has revealed that, anaerobically, the 
chief action of hot concentrated alkali upon gly- 
cogen consists of a series of degradative steps 
starting at the free reducing and of the poly- 
saccharide molecule and proceeding along the 
one short straight chain of al,4-linked glucosyl 
residues. The products formed are chiefly free 
isosaccharinic acid and a polydisperse series of 
polysaccharide acids. Quantitative measurements 
of the isosaccharinic acid split off by alkali from 
glycogen samples of known average molecular 
size, were found to be in fairly good agreement 
with the amounts expected from calculations 
based upon idealized molecules of similar size 
having the highly branched treelike structure 
which has been predicted from enzymological 
studies. Borohydride reduction of the terminal 
aldehyde group has also been found to render the 
glycogen molecule stable to alkali degradation. 
The apparent stability of "glycogen" to concen- 
trated alkali is due to the elimination of the 
reducing end of each molecule and its replace- 
ment by a saccharinic acid residue (M. R. Stet- 
ten). 



Pursuant to a study of the mechanism by 
which sugar moieties are transferred by some 
transferase enzymes several critical experiments 
have been performed. Glucose-1-0 18 , prepared 
by exchange of glucose with H 2 18 , was in- 
cubated with raffinose and levansucrase accord- 
ing to the following equation: 

glu-1-0 18 + gal-0-glu-O-fru _/ 



levansucrase / 
gal-0-glu + glu-0 18 -fru 

The sucrose formed was labeled with 18 show- 
ing that fructose is transferred as fructosyl. 
Dextran produced in the dextransucrase reaction 
was devoid of 18 showing that glucose is trans- 
ferred as glucosyl, as follows: 

dextransucrase 
n glu-0 18 -fru >- (glu)n + n fru 

These results support the prediction of Koshland 
to the effect that the transfer of an enzyme-spe- 
cific moiety occurs at the bond between the 
anomeric C atom of that moiety and the bridge 
atom. Whereas Koshland studied only hydro- 
lytic enzymes, in which water is the acceptor 
of the group transferred, the present studies are 
the first to be done on transfers from carbohy- 
drate donors to carbohydrate acceptors. It is 
concluded that a glycosyl-enzyme complex is the 
intermediate structure in these transfers: 

gl — R + Enz transferase gl — Enx + E 

(F. Eisenberg and S. Hestrin) 

Studies on the mechanism of formation of vari- 
ous mammalian mucopolysaccharides have re- 
vealed the presence of a previously unrecognized 
chondroitin sulfate polymer in human umbilical 
cord and attempts to isolate and identify it are 
in progress. In addition, it has recently been 
demonstrated that red blood cell hemolysates 
from patients with Hurler's disease are unable 
to metabolize UDPG and that the acivity is re- 
stored by freezing. The addition of magnesium, 
in small amounts, overcomes the original defect in 
this tissue and renders it unaffected by freezing. 
Further investigation of the levels of free and 
bound magnesium in normal and pathologic 
hemolysates is planned (J. Hickman). 

A study of the pattern of enzymatic degrada- 
tion of a polyuronide has been undertaken. This 
polymer, alginic acid, which contains a repeating 



156 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



unit consisting of D-mannuronic- and L-gulur- 
onic acid is utilized for growth by an unidenti- 
fied soil organism. Extracts of this organism, 
purified approximately 40-fold, catalyze the for- 
mation of a series of partially unsaturated oligo- 
saccharides leading to the formation of a new 
ketouronic acid, 2-keto-3-deoxy-6-aldehydro-hex- 
onic acid. This compound appears to be a 5- 
epimer of a similar monosaccharide recently re- 
ported elsewhere as a product of the bacterial 
utilization of hyaluronic acid and chondroitin 
sulfate. Further studies on the mechanism of the 
reaction as well as the subsequent fate of this 
metabolite are planned (J. Preiss). 

The elucidation of the structures of the vari- 
ous oligosaccharides present in human milk 
which serve as growth factors for Lactobacillus 
bifidus, and the elucidation of the reactions by 
which some of these factors are incorporated into 
the cell wall of this microorganism will be in- 
vestigated. It is felt that such an approach will 
also provide information relating to the mecha- 
nism of action of penicillin, an agent known to 
block the incorporation of N-acetyl muramic 
acid into bacterial cell walls (P. O'Brien). 

Carbohydrate Metabolism: Reactions involving 
small molecules. 

The previously described L-gulonic (DPN) de- 
hydrogenase from hog kidney has been reinvesti- 
gated and purified 100-150-fold. Studies with this 
enzyme have led to the successful isolation and 
identification of /?-keto-L-gulonic acid as an in- 
termediate in the conversion of L-gulonic acid 
to L-xylulose. The purified enzyme was shown 
to possess an unusual substrate specificity in that 
it reacts with all of the hexonic, pentonic and 
tetronic acids in which the hydroxyl group on 
the /?-carbon possesses a laevo configuration. In 
addition, the /?-keto function of acetoacetate and 
2,3-diketo-L-gulonate has been shown to be re- 
duced stereospecifically with the resultant forma- 
tion of L(+)/?-hydroxybutyrate and 2-keto-L- 
gulonate respectively. Neither of the latter two 
compounds had been previously implicated in 
mammalian metabolism. A specific decarboxylase 
acting upon /?-keto-L-gulonate has been purified 
from guinea pig liver acetone powder and L- 
xylulose identified as the unique product of this 
reaction (J. D. Smiley and J. Winkelman). 

An investigation of the enzymatic reactions 



involved in the formation and metabolism of L- 
ascorbic acid in mammalian tissues has been com- 
pleted. The enzymatic utilization of vitamin C 
has been shown to involve an oxidation to diketo- 
L-gulonic acid which is decarboxylated to yield 
L-xylonic and L-lyxonic acid. L-xylonic acid in 
turn is converted to the 5-carbon analogue, L- 
erythroascorbate and this in turn oxidized and 
decarboxylated to yield L-threonic- and L-ery- 
thronic acid (G. Ashwell, J. Kanfer, and J. J. 
Burns). 

Biosynthesis of GDP-L-Fucose. 

The pathway of the biosynthesis of this nu- 
cleotide sugar has been studied and a novel in- 
termediate, GDP-4-keto-6-deoxy-Z>-mannose, has 
been implicated in the biosynthetic conversion of 
GDP-Z>-mannose to GDP-Z-fucose. In addition 
GDP glycero-Z>-mannoheptose has been isolated 
and identified from yeast (Dr. V. Ginsburg). 

Nucleic Acids. 

Secondary structure, due to hydrogen bonding 
between specific base pairs is of increasing im- 
portance in this field. In protein biosynthesis 
it is believed that small polynucleotides with at- 
tached amino acids go to specific places on the 
RNA template, and thus the amino acids are 
lined up in an ordered way (adaptor hypothesis). 

Several recent projects are concerned with hy- 
drogen-bonded complexes between oligonucleo- 
tides and polymers. This continues work begun 
last year. The deoxynucleotide, (d) pApApA 
forms a complex with the ribose polymer, poly 
U. This is a model for the manner in which 
DNA could complex with RNA and control its 
synthesis. The effect of noncomplementary bases 
was investigated. Thus, pApApApApU bonds 
with poly U so that every "U" of poly U com- 
plexes with every "A" of the oligonucleotide. 
The terminal, noncomplementary "U" of pApA 
pApU apparently rotates out of the way (M. N. 
Lipsett, L. A. Heppel and D. F. Bradley). Re- 
sults like these are significant in assessing effect 
of mutation and abnormal bases in nucleic acid 
helices. 

The problem of nucleotide sequence, the pri- 
mary structure of the RNA chain, becomes ever 
more pressing. More and better enzymatic re- 
agents are needed for its solution. It was dis- 
covered here that alkaline phosphomonoesterase 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



157 



is the best available agent for specific removal of 
terminal phosphate from RNA and other poly- 
nucleotides. With its help the Whitfeld stepwise 
degradation method has been applied for nu- 
cleotide sequence in S-RNA. However, traces 
of nuclease still contaminate the enzyme and 
further purification is necessary (R. J. Hilmoe 
and D. R. Harkness). Studies on specificity of 
Staph, aureus nuclease, wheat germ nuclease and 
spinach ribonuclease were carried out — all for the 
purpose of using them as reagents in elucidating 
RNA structure (L. A. Heppel). 

Another investigation seeks to find out how big 
a polynucleotide has to be before it takes on the 
properties of a polymer such as nucleic acid. 
That is, at what stage is a secondary structure, 
involving hydrogen bonding between bases, ac- 
quired? The evidence is that a hexanucleotide 
can acquire such an ordered, secondary structure 
(M. F. Singer, L. A. Heppel, G. Rushizky and 
H. A. Sober). 

Studies on the mechanism of action of poly- 
nucleotide phosphorylase are being continued. 
New results on the primer requirements have 
just been obtained (M. F. Singer). Biosynthe- 
sis of RNA in a fraction from E. coli (A. L. 
Stevens) showed an apparent requirement for 
DNA. 

Studies on the structure of polynucleotides 
have been continued using, primarily, infrared 
spectroscopic techniques. Evidence that the I + 
C polymer may have a DNA-like structure has 
also been obtained (T. Miles). 

Steroids 

Studies on mechanisms of hormone action. 
Several recent studies have increased our under- 
standing of how hormones are able to exert an 
effect on metabolism by altering the activity 
of enzymes. Thus, certain estrogenic steroid 
hormones inhibit a key enzyme — glutamic dehy- 
drogenase. It was found that their ability to 
inhibit crystalline beef liver glutamic dehydro- 
genase is due to their ability to alter the struc- 
ture of the enzyme by dissociating it into sub- 
units. The subunits possess a different catalytic 
activity, alanine dehydrogenase, from that of the 
parent tetramer. The conversion of the tetramer 
to the subunit is catalyzed by steroid hormones 
and the reverse reaction is facilitated by adeno- 



sine diphosphate (G. M. Tomkins and K. L. 
Yielding) . 

Aldehyde Dehydrogenase. A steroid-sensitive 
aldehyde dehydrogenase has been discovered and 
studied. It is strongly inhibited by such com- 
pounds as progesterone and stimulated by diethyl- 
stilbestrol, cortisone and several steroids. This 
is the only case of an enzyme whose activity has 
been altered in several directions by different 
steroid hormones. The role of this enzyme in 
galactose metabolism has also been studied (E. S. 
Maxwell). 

The cytoplasmic fraction of liver contains two 
DPN-linked aldehyde dehydrogenases. Only one 
of these is steroid sensitive. Whereas proges- 
terone and the androgens inhibit this activity, the 
estrogens, and to a lesser extent cortisone, in- 
hibit when the substrate concentration is rate 
limiting, but stimulate when the substrate con- 
centration is nonrate limiting. The mechanism 
of inhibition appears to be different from that 
of stimulation. The dual nature of the estrogen 
effect may represent a prototype for a homeo- 
static mechanism by which accelerated metabolism 
occurs when substrate is present in excess and 
decelerated metabolism occurs when substrate is 
limiting (E. Maxwell and Y. Topper). 

In light of the recent finding that vitamin A 
acid can substitute systemically for other vita- 
min A forms, it was of interest to find that 
vitamin A aldehyde is irreversibly oxidized to 
the acid by aldehyde dehydrogenase, and that the 
K m for the aldehyde is about lO 6 M. The kidney 
enzyme is quite sensitive to estrogens, and this 
may account for the fact that female kidney 
contains much less of the vitamin than does the 
male kidney (T. D. Elder and Y. Topper). 

In continuation of studies on galactose metabo- 
lism, it has been found that stimulation of the 
epimerase reaction leads to acceleration of the 
transferase reaction in hemolysates (T. D. Elder, 
S. Segal, and Y. Topper) . 

Metabolism of Steroids. It has been shown 
that the liver represents a heterogenous cell popu- 
lation with respect to the steroid 4-5 double bond 
reductastes (R. F. Bakemeier and G. M. Tom- 
kins). This finding may be related to current 
ideas about antibody production in reticuloendo- 
thelial systems. In other work, it was found that 



158 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



there are at least nine separate steroid 4-5 unsatu- 
rated /J reductases (A. N. Weinberg and G. M. 
Tomkins). These are TPNH-dependent enzymes 
catalyzing an irreversible reaction. Some of 
their physical properties have been studied. They 
have similar, but not identical, mobility on di- 
ethylaminoethyl cellulose chromatography and in 
density gradient centrifugation. The pH optima 
are different and they have a different substrate 
specificity. 

Regulatory Mechanisms and Hormones 

In attempts to delineate the site of the dia- 
betogenic action of mannoheptulose (MH) the 
following observations have been made. This 
seven-carbon sugar does not affect the insulin 
effect on glucose uptake by isolated rat dia- 
phragm; thus, the peripheral action of insulin 
is unaltered by MH. The pattern and rate of 
I 131 -insulin degradation by perfused rat liver is 
unchanged by MH, indicating that the hormone 
is not more quickly destroyed in the presence of 
this sugar. A rise in blood ketone bodies is 
caused by MH even when glucose is simultane- 
ously injected. Sedoheptulose, an isomer of 
mannoheptulose, does not elicit these effects. It 
has been tentatively concluded that the site of 
diabetogenic action of MH is the pancreas. A 
gluconogenic effect of MH appears to be medi- 
ated through the adrenals (E. Simon, R. Scow 
and S. Chernick). 

The nature and locus of binding of insulin by 
target tissues, and the characteristics of the en- 
zymic degradation of this hormone by mammal- 
ian liver are under investigation. 

Chemical and electrophoretic data obtained 
with a fluorescent derivative of insulin indicate 
that the product contains 1.25 fluorescin radicals 
per molecule of protein. Results of rabbit bio- 
assays indicate that the derivative possesses y 2 
to % of the activity of the native hormone. Pre- 
liminary experiments involving the use of fluo- 
rescence microscopy have demonstrated that the 
derivative is strongly bound by leucocytes but 
not by erythrocytes (F. Tietze and G. Morti- 
more) . 

Following treatment of insulin-I 131 with highly 
purified liver "insulinase" approximately 15 per- 
cent of the total radioactivity of the substrate 
becomes soluble in trichloroacetic acid in contrast 
to nearly 100 percent with crude liver homogen- 



ate. This result may indicate that the purified 
preparation represents only one component of a 
complex that constitutes, in its entirety, the en- 
zyme system known as "insulinase" (H. Katzen 
and D. Stetten). 

Oxytocin and vasopressin stimulate glucose 
oxidation by mammary gland in vitro. The ef- 
fects of these hormones are additive to that of 
insulin, but not to each other (T. Goodfriend, 
J. Cohen, and Y. Topper) . 

The chemical nature of the humoral agents 
which accumulate during uremia and the chemi- 
cal basis for the changes in cerebral function in 
uremia are being studied. It has been found 
that normal rat brain cells, when suspended in 
serum from uremic patients, generate signifi- 
cantly greater yields of C 14 02 from glucose-C 14 
than do the same cells suspended in normal 
serum. This finding provides an assay for an 
agent in human uremic serum which alters the 
metabolism of nervous tissue. Attempts to con- 
centrate and ultimately to identify the agent or 
agents responsible are being pursued. If this 
goal is achieved a rational therapy for uremia 
may result (N. Cummings, D. Stetten). 

Pyridine Nucleotides and Other Coenzymes 

A number of TPNH-dependent enzymes have 
been shown to be inhibited by the end product, 
TPN. The biological significance of this finding 
has been explored and it is felt that this inhibi- 
tion, rather than being fortuitous, represents an 
important biological control ( G. Ferro-Luzzi and 
G. M. Tomkins) . 

In continuation of studies designed to study 
the metabolic fate of intracellular^ generated 
reduced pyridine nucleotides the synthesis of 
cholesterol and fatty acids by rat liver slices has 
been investigated. The synthesis of cholesterol 
(digitonin-precipitable steroids) from acetate- 
2-H 3 ,C 14 results in a product with an isotopic 
ratio of H 3 /C 14 of 0.4. This is in strikingly good 
agreement with that derivable fro mtheoretical 
considerations, namely 0.44 ± 0.04. Similar ex- 
periments utilizing acetate-2-H 3 ,C 14 incorporation 
into fatty acids revealed an isotope ratio of 0.24, 
to be contrasted with a theoretical value for 
stearic acid synthesis of 0.11. Which of the con- 
siderations utilized in calculating the theoretical 
value for stearic acid is in need of modification 
has not been determined. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



159 



The metabolism of TPNH and DPNH gener- 
ated intracellular^ has also been studied. This 
was accomplished with the use of glucose-1-C 14 , 
H 3 and glycerol-2-H 3 as substrates. It has been 
shown that these two reduced pyridine nucleo- 
tides are not equivalent in the cell. TPNH is 
utilized in fatty acid and cholesterol synthesis to 
an extent 10 to 20 times greater than DPNH. 

Utilizing the H 3 /C 14 ratios in fatty acids and 
cholesterol from glucose-6-C 14 ,H 3 and acetate-2- 
C 14 ,H 3 an estimate has been made of labilization 
of hydrogen in the Embden-Meyerhof pathway. 
The results indicate that % of the hydrogens 
bound to C-6 of a molecule of glucose are ex- 
changed during conversion to acetyl coenzyme A. 
Presumably this occurs at the phosphoenolpyru- 
vate-pyruvate reaction (B. Bloom and D. Foster) . 

The biosynthesis of thiamine and its phos- 
phorylated derivatives by baker's yeast is being 
investigated. An enzyme which catalyzes the 
condensation of the two-ring moieties of thiamine 
phosphate according to the following equation: 

Mg+ + 
"Pyrimidine"-PP + "Thiazole"-P ===== 
Thiamine-P + PP 

has been isolated and purified about 1,000-fold. 
The reaction has an equilibrium constant of ap- 
proximately 10, and is inhibited by pyrophos- 
phate noncompetitively. Procedures have been 
devised for the synthesis of hydroxymethyl pyri- 
midine and the corresponding mono- and pyro- 
phosphate derivatives (I. Leder). 

Protein and Amino Acid Synthesis 

Cell-free extract which makes small, but ap- 
parently significant, quantities of the inducible 
enzyme penicillinase from B. cereus has been 
obtained. The requirements for this system are 
under study at the moment (M. Nirenberg) . 

Histidine Biosynthesis 

The first step of histidine biosynthesis in 
Salmonella has been shown to be the reversible 
condensation of phosphoribosyl-pyrophosphate 
and ATP to form N-l-(5'-phosphoribosyl)-ATP 
and pyrophosphate. The condensation product 
has been isolated and its structure proved. The 
enzyme catalyzing the reaction has been described 
and named phosphoribosyl-ATP pyrophosphory- 
lase. It is lacking in one class of histidine mu- 



tants. The enzyme is strongly inhibited by histi- 
dine. None of the other enzymes of the pathway 
is inhibited by histidine and the inhibition of the 
first enzyme by the end product seems to be an- 
other example of feedback inhibition control. 

In addition to feedback inhibition another 
mechanism, repression, is capable of regulating 
the amount of histidine synthesized in Salmo- 
nella. Repression results in control of the rate 
of synthesis of the enzymes of a pathway by the 
end product of the pathway (B. N. Ames and 
R. G. Martin) . 

Enzymatic Utilization of Model Compounds 

Continuing studies on the mechanism of en- 
zyme catalyzed aldehyde oxidation have resulted 
in information concerning the sites of substrate 
binding to protein. Employing techniques of en- 
zyme digestion and competitive inhibition, it has 
been concluded that aldehyde substrates are 
bound to closely juxtaposed SH groups of the 
enzyme, whereas pyridine nucleotides are bound 
at sites other than sulfhydryl groups. The fact 
that each of 10 different aldehyde dehydrogen- 
ases tested are sensitive to inhibition by arsenite, 
a specific inhibitor of disulf hydryl systems at low 
concentration, strengthens the case of disulf- 
hydryl involvement for enzyme catalyzed alde- 
hyde oxidation. The precise manner in which 
such sulfhydryl groups participate is in the 
realm of speculation but probably involves a 
series of disulfide interchange reactions. 

An enzyme thiooxidase has been purified from 
a mold, Piricularia oryzae, which catalyzes the 
oxidation of conjugated sulfhydryl compounds. 
The enzyme catalyzes the oxidation of catechols 
as well and is analogous in many respects to 
polyphenol oxidases. Because the thiol substrate 
is oxidized to a disulfide and no further, thus 
differing from polyphenol systems which oxidize 
catechol extensively, the mechanism of the oxida- 
tion may be studied without interference from 
competing reactions. Here, too, are indications 
for the involvement of closely linked sulfhydryl 
groups (W. B. Jakoby, and G. Aurbach). 

Laboratory of Physical Biology 

The Laboratory of Physical Biology comprises 
five sections which devote attention to basic re- 
search in disciplines varying from physics and 



160 



ANNUAL, REVIEW OF INTRAMURAL RESEARCH 



chemistry to physiology and often cooperative 
ventures involving several disciplines. In a gen- 
eral sense, the several projects are related by the 
attention given to the material aspects of life 
processes, but specifically with the energetic and 
structural interconversion which is responsive to 
the various environmental factors, both immedi- 
ate and remote, that interact with organisms. 
Much cooperative work was carried out with 
personnel of other Institutes and some at labora- 
tories made available in off campus research 
centers. 

In the area of photobiology studies on the 
basic phenomena of photosynthesis are concerned 
with the primary step in photomechanisms. 
Early transient phenomena are of special inter- 
est. Dr. Olson has developed a spectromicro- 
photometer for observing fluorescent spectra and 
absorption changes at half second intervals. The 
transient formation of a yellow fluorescent com- 
ponent in what (max. emission 540 m^) has been 
demonstrated to be nonenzymatically produced, 
to require interface distribution, and to depend 
on availability of oxygen. The process is seen 
to be reversible and similar to others in activated 
components of protochlorophyll and numerous 
porphyrins. 

Photoreception in animal tissues has been 
shown by Dr. Hagins to result in the release of 
selectively concentrated potassium ions in the 
receptor cells. This is accompanied by an elec- 
trical impedance fall which is subject to sup- 
pression by procaine but without effect on the 
externally observed action current. A model has 
been devised which accounts for most of the find- 
ings in terms of known properties of electrically 
excitable cells. 

The effects of destructive radiation have been 
studied by Dr. Greenblatt, who, in cooperation 
with Dr. Elkind of the Cancer Institute, has 
shown that clonal techniques of studying the 
survival and the changes in chromosome prop- 
erties can be used to great advantage in cell cul- 
tures of the Chinese Hamster. A model was 
devised which demonstrates critical points in the 
phenomena of chromosomal injury which are 
subject to further experimental proof by the 
techniques devised in this study and in coopera- 
tion with Dr. Royce Lockhart. 

By means of nuclear magnetic resonance 
(NMR) and electron paramagnetic resonance 



(EPR), Dr. Becker has shown the molecular 
structure of a number of substances of biological 
interest in many cooperative studies performed 
with scientists of both NIH and nongovernmen- 
tal institutions. Information obtained from the 
techniques of NMR and EPR supplements that 
obtained by infrared spectroscopy and provides 
detailed information on structure, particularly 
on molecular interactions and the character of 
molecules containing impaired electrons, includ- 
ing free radicals and paramagnetic atoms and 
ions. A substantial addition to the number of 
accurately determined hydrogen bond energies 
now in the literature has been made. This work 
has thrown support to some tentative theories 
and doubt on other long held generalizations 
regarding hydrogen bonding. Studies of por- 
phyrins, of interest to Dr. Watson (University 
of Minnesota) have provided information on the 
position of functional groups and tautomerism 
of the NH protons in certain metal-free por- 
phyrins. A free radical intermediate in the oxi- 
dation of chlorpromazine in vitro is being studied 
to elucidate its apparent second order decay 
reaction demonstrated by the methods used in 
this project. Many other NMR spectra have 
been made for other investigators and found 
helpful and even instrumental in solving chal- 
lenging structural problems. 

The use of dipole moment, dielectric polariza- 
tion and spectroscopic absorption, rotatory dis- 
persion and dichroism by Dr. Charney has re- 
sulted in several different methods of elucidating 
the role which intermolecular forces play in 
molecular organization . The importance of 
atomic polarization rather than molecular char- 
acteristics in causing the apparent dipole moment 
of p-quinone was demonstrated. The binding of 
protoporphyrin to Bovine serum albumin was 
studied to elucidate the control of the stoichi- 
ometry of protein binding by copper. Interest 
has been developing in a method of studying 
polar molecules in the glycerol-water-salt solution 
glass formed at liquid nitrogen temperature. 
Nucleosides exhibit characteristic and specific 
differences in the sharpening of the spectral 
bands under these conditions. In addition, the 
effect of solvents on spectral absorption has been 
shown as a function of Van der Wall's constant 
"a" and related to the ratio of dipole moment 
and polarizability. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



161 



Studies of steroid photo reactions have been 
continued by Dr. Brackett with Dr. Sharpless 
to show the quantum requirements for ergosterol 
transformation. The data are consistent with 
those of Havinga and give in addition values at 
three longer wavelengths. The work also pro- 
vides further information on the time course of 
transformations involving tachysterol and pre- 
calciferol which have been the subject of previ- 
ous studies. The application of analog computer 
techniques with the assistance of Mr. Hahn has 
facilitated the otherwise tedious reduction and 
analysis of data so that much more information 
may be brought to bear on the proof of current 
concepts. Instrument development in all areas 
has been directed toward automation and com- 
puter analysis. 

Observations of the optical rotatory dispersion 
in the ultraviolet region by Drs. Weiss and Char- 
ney suggest that the Cotton effects of ergosterol 
and its stereo isomers cannot be explained solely 
through action of the asymmetric carbon atoms 
adjacent to the dienic chromophore, implying a 
contribution by the latter and thus a deviation 
from planarity in the dienic system. 

The application of flash photometry to ergos- 
terol studies by Dr. Adams has shown that ther- 
mal reactions are of importance in the elucidation 
of the complex isomerizations which take place 
in irradiation by various means. 

Dr. Ziffer has shown that a differential assay 
of vitamins D 2 and D 3 is available through the 
finding that these products are transformed to 
their pyro- and isopyro-calciferol isomers respec- 
tively during gas chromatography. 

In the past year the formation of a new section 
studying the effects of high energy radiation on 
substances of biological interest and on organ- 
isms themselves has proceeded slowly with the 
difficult problem of setting objectives with feasi- 
ble modes of attack. Under the direction of Dr. 
Liddel the means for thermal neutron exposure 
were devised by Dr. Malich in the installation 
of a 400 Kev positive ion accelerator which is 
currently able to yield well over 100 times the 
product of the Polonium-Beryllium source avail- 
able earlier. Calibration of the neutron flux has 
been carried out in such a manner as to give ab- 
solute values of neutrons per unit volume at vari- 
ous positions in the exposure chamber. The use 
of this source for exposures both of substances 



of biological interest and living organisms is 
currently being arranged. 

In anticipation of other objectives of this sec- 
tion, Dr. Kempner has continued his studies of 
radioactively labeled compounds and their syn- 
thesis in organisms such as yeast cells. His 
studies with uracil and fluorouracil show that 
both are incorporated only into RNA and not 
into DNA. This allows the use of fluorouracil 
in the study of the selective action of the cells 
between these two compounds at various stages 
of synthesis of RNA. In comparison with pre- 
vious work using other analogs in protein syn- 
thesis, it is seen that the nucleic acid synthesis 
mechanism has the ability to select pyrimidines 
in at least three observable steps. 

The use of controlled low voltage electrons to 
localize various functional sites in large mole- 
cules has been exploited by Dr. Preiss. He has 
shown that basic globular proteins which have 
been subjected to dry, in vacuo bombardment 
acquire the ability to induce an excess turbidity 
in polynucleotide solutions. This turbidity is a 
non-monotonic, protein characterizing function of 
the dose and an analytical treatment of the data 
has been made correlating experimental exam- 
ination of the significance of primary, secondary, 
and tertiary structures. The importance of iso- 
electric pH and molecular weight of the pro- 
teins has been shown and the importance of the 
number of S-S bridges is under study. The gen- 
eral method promises much application in an 
area otherwise singularly inaccessible to quan- 
titative assessment. The comparative effects of 
thermal and faster neutron radiations are under 
study. 

Studies in physico-chemical aspects of semi- 
permeable membranes have been pursued by Drs. 
Sollner, Caplan, Shean and James with the find- 
ings that self-exchange and allo-exchange of ions 
can be very satisfactorily described by current 
theoretical treatments devised here. A new ap- 
proach in the study of perm-selective cation ex- 
change membranes has centered on the pro- 
nounced frequency dependence of the resistance 
of such membranes. In the case of highly 
charged membranes, new light has been thrown 
on the anomalies which classical and current 
theory cannot resolve. The inclusion of the "con- 
tractile effect" of polyelectrolytes in such con- 
siderations can improve the rationalization of 



162 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



procedures which otherwise give such anomalous 
results. Studies on nonstructural, homogeneous 
("oil") membranes are underway showing much 
greater ionic selectivities and reaction rates than 
previously described. 

In another area of Molecular Biophysics the 
investigation of macromolecular organization of 
substances of biological interest is under study 
through electron microscopy and X-ray diffrac- 
tion by Dr. Labaw and Mr. Mosely. They have 
shown that in the study of fine structure it is 
possible to see striated images from periodic 
objects in which the period may be one-half, 
equal, or twice the actual period of the object. 
Definition of the conditions producing such phe- 
nomena has been accomplished for some organic 
substances in an attempt to facilitate the cor- 
rect interpretation of such fine structure images, 
particularly those near the limit of resolution. 

Studies in Physical Biochemistry have contin- 
ued the analysis of the various phenomena con- 
cerned with the relationship of structure and 
function at the molecular, cellular, and organ 
level. Dr. Laki and his group continue to ana- 
lyze the mechanical, chemical, and energetic fac- 
tors in the special protein complexes that account 
for the unique behavior of muscle, blood clotting 
and a number of allied systems in which the inter- 
conversion of energy and structure takes place 
through highly specialized properties of discrete 
parts of oftentimes huge molecular aggregates. 
Muscle is one example of a number of biological 
transducers which has received much study here 
but other aspects such as the mode of action of 
enzymes are actively being pursued as in the 
case of thrombin, carboxypeptidase, A, B, myo- 
sin ATPase, and others. In addition, the phe- 
nomena of the polymerization of proteins has 
remained of interest since it lends insight on the 
formation of networks which merge into the 
visible structures which can be directly observed. 

It was found by Dr. Bowen that in glycerol 
treated muscle fiber ADP and phosphate ion 
suppress the work done by fiber-bundles in ATP 
induced contractions but that this is less effec- 
tive than the suppression of ATP splitting by 
homogenized fiber. He also showed that the 
diffusion of ATP in fiber bundles could be 
determined by measuring the extra tension de- 
veloping in various sized bundles of ATP tensed 
muscle on the addition of ADP. 



Studies on myosin by Dr. Carroll show that 
free diffusion measurements of dog heart myosin 
correspond to inhomogenious material in both 
normal as well as failing hearts and the deduced 
molecular weight (5.0 x 10 5 ) must therefore be 
considered an average value. Dr. Laki found 
that tryptic digestions of myosin showed "finger- 
prints" in which the count of arginine containing 
spots gave an estimate of a minimal molecular 
weight in the neighborhood of 2.0 x 10 5 . Such 
a weight indicates multiple aggregation into 
larger myosin molecules through orderly side 
to side and end to end associations. 

Drs. Stewart and Bowen showed by labeling 
techniques that inorganic phosphate binding is 
present in myosin ATPase but find that it is 
probably not a contributing factor to the high 
initial rate of action. 

The dissection of myosin into three distinct 
components by specific chemical reagents has 
been used by Drs. Kominz and Carroll in study- 
ing the mode of synthesis and structure of this 
protein. Comparative studies are in progress. 

In studies on F-actin, Dr. Laki has found by 
using labeled ATP that F-actin binds only one 
ADP molecule but a varying number of inor- 
ganic phosphate molecules depending on the con- 
centration of phosphate in the medium. 

The equilibrium constant for dimerization of 
human mercaptalbumin with mercury or mer- 
curials was shown by Dr. Simpson to have a 
value two orders of magnitude smaller than for- 
merly estimated and that this presented an 
interference problem in reactions with other ca- 
tions. 

In the study of fibrin-fibrinogen transforma- 
tion, Dr. Gladner, in cooperation with Dr. Folk 
of NIDE, showed that the complete sequence of 
amino acids in peptide B of co-fibrin is: N- 
acety . thre . gly . phe . pro . asp . tyr . So* . asp . 
glu . gly . gly . asp . asp . arg . pro . lys . val . 
gly . leu . gly . ala . arg. 

They also have shown that very highly purified 
carboxypeptidase B from pig pancreas has a 
molecular weight of 34,000 and that it contains 
one gram atom of zine per mole of the enzyme. 
It consists of a single polypeptide chain. Dr. 
Carroll showed that pyruvate and a-ketoglutarate 
dehydrogenation complexes from Escherichia coli 
are large multienzyme units of 4.8 and 2.4 mil- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



163 



lion grams, resp., capable of carrying out a series 
of reactions involving several coenzymes. 

In the field of amino acid analysis a new ap- 
proach has been successfully made by Dr. Saroff 
in collaboration with Dr. Karmen of NHI by the 
adaptation of gas chromatography. The key to 
this success was the preparation of N-trifluoro- 
acetylmethyl esters of the amino acids. The 
method has yielded good results with valine, 
methionine, serine, glutamic acid, phenylalanine, 
hydroxyproline and cystine. 

Drs. Irreverre and Viswanatha have estab- 
lished the presence of hydroxylysine in trypsin 
and also detected it in chymotrypsin. Dr. Irre- 
verre found that an interesting a-amino acid, 
homocitrulline, was present in the urine of in- 
fants. It is not found in adults or older chil- 
dren. 

Physiological studies which range from mam- 
malian species through invertebrates form a 
varied approach to the effects of environmental 
and other physical factors on biologic functions. 
A continuation of Dr. Park's investigation of the 
cyclical phenomenon of gonadal development in 
fresh water Hydra shows that it is independent 
of population density over a 25-fold range but 
budding is inversely related to crowding. The 
effect of environmental factors either directly or 
via secondary mechanisms is being further in- 
vestigated. 

Studies on the biochemical transformations 
during insect metamorphosis by Dr. Levenbook 
have shown that blood citrate titers found in 
larvae of 17 species are high before metamor- 
phosis but fall steeply during this stage only to 
increase to a peak during mid-pupal life before 
the decline to adult levels. Dr. Buck has shown 
by studies of response latency that the neuro- 
effector linkage of the firefly lantern has three 
steps in electrical stimulation. This is viewed 
as involving a conventional neural conduction at 
adequate stimulation levels; an excitation of an 
intermediate unit at higher stimuli, probably the 
tracheal end-cell, which can be considered ana- 
logous to the motor end plate of muscle; and, 
third, a direct excitation of the photocyte. Dr. 
Keister has shown that blowfly adults are able 
to maintain respiration at hypoxic levels lower 
than possible for larvae or pupae but that in all 
stages temperature and hypoxia influence both 



normal and basal metabolism in a parallel man- 



ner. 



Studies on the effect of hypoxia in higher 
forms have been carried on by Dr. Altland as in 
the past and are directed to the effects on the 
blood and circulatory tissues. In collaboration 
with Dr. Highman, it has been shown that hy- 
poxia plays a recognizable role in the experi- 
mental production of pulmonary arteriosclerosis 
and in calcium deposition in atherosclerotic le- 
sions in the aorta and pulmonary vessels. These 
lesions are correlated with very high serum 
cholesterol values in cholesterol-fed rabbits ex- 
posed to high altitudes. Altitude tolerance stud- 
ies in chickens show that they have a lower 
tolerance than any small warm blooded animals 
previously studied. The altitude exposure of 
dogs results in an increase in certain serum en- 
zymes beyond normal levels revealing a hitherto 
unexplored physiological mechanism involved in 
hypoxic stress. In conjunction with Dr. F. 
Smith, NCI, it was discovered that there is an 
increase in the titer of antisheep erythrocyte 
hemolysin in rats exposed to high altitude and 
it is felt that this may provide a new experi- 
mental method for increasing other antibody 
titers. 

Studies on the circulatory reaction of sensitive 
mammalian species to plasma expanders by Dr. 
L. Marshall have shown that moderate doses of 
dextran increase the incidence of fatal insulin 
convulsions in rats while a larger dose is protec- 
tive. A relation to the significant hyperglycemia 
found in the latter under conditions of conscious- 
ness may be inferred. During pentothal anes- 
thesia such a hyperglycemia is greater than in 
conscious rats and this coupled with the reported 
finding that high blood glucose suppresses dex- 
tran reactions, and the previously found vaso- 
depression enhancement through pentobarbital 
may explain why the lack of demonstrable re- 
actions to dextran are seldom seen under anes- 
thesia in rats. It was shown that after insulin, 
vasodepression followed intravenously adminis- 
tered dextran in rats but less severely than in 
normals. Similar relations are observed in edema 
formation, but hematocrit values show that after 
insulin treatment there may be greater general- 
ized fluid loss to the tissues than in the so-called 
"target areas" in which it seems to be reduced. 
In dogs receiving: insulin there is seen an at- 



164 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tenuated reaction to polyvinyl-pyrrolidone 
(PVP) given intravenously. Under pentobar- 
bital anesthesia, the rise in blood sugar following 
PVP injection is no greater than when conscious, 
correlating with earlier findings that this anes- 
thesia introduces no alteration in reaction. 

The section has also continued some work on 
pulmonary physiology and currently Dr. Specht 
and Mr. Brubach are studying the use of heavy 
gas mixtures for demonstrating the efficiency of 
lung ventilation and in a new application of 
Archimedes principle for determining the den- 
sity of the body by this heavy atmosphere. The 
latter method is alternative to and perhaps more 
sensitive than current clinical procedures. 



Laboratory of Chemistry 

Medicinal Chemistry 

Work has continued on the synthesis of anala- 
gesic drugs. The analgesic drug Phenazocine 
(NIH 7519) developed in recent years through 
the work of this section is now in clinical use for 
injection as Prinadol (Smith, Kline & French 
Labs.) in this country and as Narphen (Smith 
& Nephew, Ltd.) in England. Studies in our 
laboratories indicate a broad spectrum of clinical 
utility for oral preparations and marketing of 
an oral form seems imminent (Drs. Eddy, Co- 
chin and May). 

The benzomorphan class of compounds con- 
tinue to show high analgesic potency and low 
physical dependence capacity in monkeys. One 
of the more interesting benzomorphans under 
study has shown excellent carryover of pain-re- 
lief from mouse to man. In this series, com- 
pounds in which the 5 and 9 alkyl groups are 
trans (equatorial-equatorial), are at least 10 
times more active than the much more readily 
obtainable eis-counterparts (Drs. N. B. Eddy and 
E. L. May). 

A new and improved synthesis (Dr. E. M. 
Fry) of the benzomorphan class of neurophar- 
macologic agents has been developed. This syn- 
thesis, a 3-step sequence starting from pyridine 
alkohalides, is based on the 1.2-shift of the ben- 
zyl or substituted benzyl group of 1,2,5,6-tetra- 
hydropyridine quaternaries (Stevens rearrange- 
ment) and is particularly applicable to 4-substi- 



tuted and unsubstituted pyridines not otherwise 
practicably amenable to the benzomorphan syn- 
thesis. By this new method and by the Grewe 
procedure, a new (5,9-diethyl) benzomorphan 
and its diastereoisomer at C-9 (the latter in min- 
ute yield) have been synthesized from 3,4-diethyl- 
pyridine (Dr. E. L. May and Mr. J. H. Ager). 

There has been further demonstration of the 
importance of electrical effects in the stereo- 
chemical control of addition of H-H and CH 3 -II 
to the carbonyl group of 9-oxobenzomorphans. 
Either possi^e diastereoisomeric carbinol. to the 
exclusion of the other, may be obtained depend- 
ing on whether the proximate nitrogen is quarter- 
nary or tertiary (-). These carbinols (analogs 
of 14-hydroxydihydrocodeinone and morphinone) 
and particularly their 0-acetyl derivatives are 
potent analgesics (Dr. Seiichi Saito and Dr. E. L. 
May). 

Codeine has been transformed by an interest- 
ing sequence of reactions into an analog of 
phenazocine containing a morphine-like oxygen 
bridge. Continued research on the analogs (at 
the ester function) of acetyl choline has revealed 
that when nitrogen is diphenethyl substituted, 
maximum analgesic activity is seen with the 
acetyl. In a 5-step synthesis an analog in which 
one of the N-phenethyl groups was replaced by 
p-AcOC 6 H 4 COCH 2 was obtained, although its 
isolation was complicated by its remarkable sen- 
sitivity to solvolytic cleavage (Dr. J. G. Mur- 

phy)- 

In the immunochemical field vinyl glucoside 
(via the Hofmann elimination reaction) and p- 
hydroxystyrene have been synthesized and pre- 
liminary polymerization and copolymerization 
studies performed. It is believed that such poly- 
mers and copolymers may serve as the backbone 
for attachment of determinant groups in the total 
syntheses of antigenic substances (Mr. T. D. 
Perrine) . 

It has been found that thyroxin will signifi- 
cantly depress N-demethylase activity when given 
once a day at 0.5-0.75 micrograms per ki'ogram 
of rat over a thirty day period and 4 mg./kg. 
of morphine administered twice daily for one 
week results in significant reduction of N-de- 
methylase activity. The longer the drug is given, 
the lower the dose at which one sees significant 
reduction (Dr. J. Cochin). 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



165 



Carbohydrates 

Further studies of the sugars in the avocado 
and Sedum species have shown that, in addition 
to the D-glycero-D-manno-octvAose reported ear- 
lier, the avocado contains a second octulose and 
also two nonuloses. Sedum. extracts also appear 
to contain a second octulose and the same two 
nonuloses. Nine-carbon sugars have not hereto- 
fore been encountered in nature (Drs. Richt- 
myer and Sephton). 

Condensation of products formed from gly- 
cosides by periodate oxidation, followed by re- 
duction, yields the glycosides of aminosugars. 
By this route kanosamine, 3-amino-3-deoxy-D- 
glucose, the sugar moiety in the antibiotic kana- 
mycin, was synthesized (Dr. H. H. Baer). 

/?-Sedoheptitol ( D-glycero-D-gluco-heptito\ ) 
has been condensed with formaldehyde and the 
structure of the resulting tri-0-methylene deriva- 
tive has been proved to be 1,3 :2,4 :5,7-tri-6>- 
methytene-D-glycero-D-gluco-hept'itol (Mr. E. 
Zisis). 

The synthesis of 1-substituted aldoses through 
the reaction between 1-thio-aldose derivatives 
and various heavy metal salts, a process discov- 
ered here last year, has been investigated from 
various aspects. One result of this work is the 
discovery of a wholly new synthesis of 3-deoxy- 
ribose nucleosides (Dr. Pedersen). 

Methods for the synthesis of labile 1-acylal- 
doses have been explored. Among other path- 
ways, that through fully benzyl ated aldofurano- 
syl halides has been investigated, with the D- 
ribofuranose and L-arabinose series as examples 
(Dr. R. Barker). 

The discovery that the disaccharide sophorose, 
2-0-/?-D-glucopyranosyl-D-glucose, is a contami- 
nant in U.S.P. dextrose and also a potent inducer 
for the formation of at least one enzyme has 
directed attention to this rare sugar. In order 
to make the substance available for biochemical 
research, a synthesis, giving the sugar in 30 per- 
cent yield, was evolved (Dr. B. Coxon). 

Metabolites 

The program on the development of nonenzy- 
matic methods for the cleavage of peptide bonds 
in proteins, peptide hormones and enzymes at 
present occupies the majority of the members of 
the Section on Metabolites. With a two-fold aim 
in mind independent methods for "auditing" 



primary sequences of proteins established by the 
"accounting method" of overlapping sequences 
of tryptic peptides have been developed and the 
selectivity of the chemical agents employed has 
been utilized to modify enzymes and proteins 
and to correlate chemical reactivity of special 
functional groups or centers with the secondary 
or tertiary structure of the protein. 

The most important discovery made during the 
year 1960 in this area was the selective cleavage 
of peptide bonds next to methionine, which was 
initially accomplished by addition of alkyl hal- 
ides to yield tertiary alkylmethionine sulfonium 
derivatives which, on heating in aqueous solu- 
tion, broke down under cleavage with release of 
an N-terminal pipetide and a peptide with C- 
terminal homoserine (lactone) (Drs. Lawson, 
Gross and Foltz). 

A major improvement was the discovery of 
cyanogen bromide as the agent of choice for 
cleaving methionine peptides at room tempera- 
ture. In this case the intermediate methionine 
cyanosulfonium salt breaks down by an intra- 
molecular process smoothly and rapidly at neu- 
tral or acidic pH to yield, in addition to the 
aforementioned peptide fractions, methyl thio- 
cyanate which was assayed by gas chromatogra- 
phy (Dr. E. Gross). 

The new methionine cleavage has been applied 
to bovine pancreatic ribonuclcase. The formation 
of a novel heptadecapeptide, a chemical tail pep- 
tide (in contrast to the enzymatic 20-residue S- 
peptide produced by the action of subtil isin) was 
observed which had the original N-terminal ly- 
sine in addition to a C-terminal homoserine 
(lactone). By electrophoretic separation and by 
dinitrophenylation technique the topography of 
the cleavage of the 4 peptide bonds next to the 
4 methionines in ribonuclease has been deter- 
mined. In the light of these new results, the 
standing controversy between the Rockefeller In- 
stitute and the National Heart Institute on the 
positions of glutamic acid and serine occurring 
either in positions 18 or 11 has been answered 
for native ribonuclease in favor of position 11 
for glutamic acid, the position favored by An- 
finsen and his group (Dr. E. Gross). 

Preliminary results on the application of cy- 
anogen bromide to the a-chain of human hemo- 
globin, which contains 2 methionines, have been 
obtained in collaboration with W. Konijjsberg 



166 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of the Rockefeller Institute and point to success- 
ful and selective cleavage (Dr. E. Gross). 

Work on the structurally extremely difficult 
cyclic antibiotic gramicidin-A, containing over 
40 percent tryptophan, has been continued and 
put on a more secure basis by the preparation 
of 60 grams of the pure gramicidin-A in col- 
laboration with the Research Laboratories of the 
Schering Corp. in Bloomfield, N.J. (Drs. S. 
Ishii and E. Gross). 

Selective oxidation of all the tryptophans in 
gramicidin and selective partial cleavage of pep- 
tide bonds next to tryptophan has been achieved 
by the use of N-bromosuccinimide in alcoholic 
aqueous solution in yields approximating 50 per- 
cent. Countercurrent distribution of the reac- 
tion mixture gave ethanolamine as the major 
water-soluble fragment and at least 3 lipophilic 
ninhydrinpositive fragments of lactonic charac- 
ter, one of which was further purified by reverse 
phase paper chromatography (Dr. E. Gross). 

In extension of the investigation on the action 
of N-bromosuccinimide on trypsinogen and its 
derivatives T. Viswanatha, LPT-NIAMD, and 
W. B. Lawson made a similar study of the ac- 
tion of N-bromosuccinimide on chymotrypsin. 
In addition to the oxidation of tryptophan resi- 
dues, one of the tyrosine residues is also affected 
by treatment of the protein with N-bromosuccini- 
mide under the conditions employed in these 
studies. The oxidative modification of the en- 
zyme resulted in a loss of catalytic activity to- 
ward typical protein and ester substrates. Such 
partially inactivated enzymes still possessed the 
ability to incorporate phosphoryl or acetyl 
groups upon reaction with diisopropyl phospho- 
fluoridate or p-nitrophenylacetate respectively. 
Oxidation of chymotrypsin with N-bromosuc- 
cinimide seems to retard both the acylation and 
deacylation steps in the catalytic process without 
affecting the K m value. A direct measurement of 
the rate of deacetylation of C 14 -acetyl chymo- 
trypsin has been made. 

Contrary to data previously reported from 
other laboratories, the oxidative splitting of ty- 
rosyl-cysteine bonds has been achieved in high 
yield. The N-bromosuccinimide method, when 
applied to S-carboxymethylribonuclease, gave 
consistently higher yields than with the intact 
enzyme. Conditions for the differential cleav- 
age of tryptophyl and tyrosyl peptide bonds 



with N-bromosuccinimide have been worked out 
for mixtures of tryptophyl and tyrosyl model 
peptides (Drs. J. G. Wilson and L. A. Cohen). 

With regard to the difficult task of utilizing 
the imidazole ring of histidine as a potential 
functional group for neighboring group effects 
that may labilize and cleave C-histidyl peptide 
bonds some progress has been made. The easily 
removable N-toluenesulfonyl group attached to 
imidazole makes it stable to oxidative degrada- 
tion. In the N-bromosuccinimide cleavage of 
S-carboxymethylribonuclease the oxidative cleav- 
age of a histidylphenylalanine bond is a possi- 
bility (Drs. Wilson and Cohen). 

A great number of model peptides containing 
hydroxyproline, an important building stone of 
collagen, have been synthesized (Dr. J. Francis) 
and investigated with regard to conditions for 
non-enzymatic cleavage. In the course of this 
investigation, a large body of data on the funda- 
mental reaction mechanisms of peptides derived 
from cyclic and open y- and "--hydroxy and un- 
saturated amino acids have been accumulated. 
The study of N-tosyl and N-benzoyl-allyl- and 
-methylallylglycine amides and peptides has con- 
tributed to an understanding of the mechanism 
of the iminolactonization reaction observed with 
N-bromosuccinimide in phosphate buffer at pH 7 
(Dr. N. Izumiya). 

As an outgrowth of the studies on hydroxy- 
proline its dehydration product 3,4-dehydropro- 
line, which is not accessible from hydroxyproline, 
has been synthesized by reduction of pyrrole-a- 
carboxamide with phosphonium iodide in fuming 
hydriodic acid. The amide of this new amino 
acid is optically unstable. This has led to an 
unprecedented combination of enzymatic resolu- 
tion with asymmetric transformation. This am- 
ide was resolved by leucine amino peptidasee of 
hog kidney. Ammonia was liberated from the 
amide and the optically active L-amino acid was 
formed in yields substantially greater than the 
theoretically possible 50 percent of L-isomer. 
Evidently yields of 100 percent of L-isomer were 
formed which under the conditions of enzymatic 
resolution partially reverted to racemic material. 
This type of resolution presents the kind of 
model which has been discussed but never real- 
ized in theories dealing with the origin of life 
and optically active matter in nature where nor- 
mally 100 percent of L-amino acids, but as a 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



167 



rule no D-forms, exist (Dr. A. V. Robertson). 

In collaboration with the Laboratory of Clini- 
cal Biochemistry, NHI, a new spectrophotometry 
assay method for L-amino acid oxidase from 
snake venom has been developed based on the 
quantitative conversion of 3,4-dehydroproline to 
the strongly absorbing pyrrole-2-carboxylic acid 
by this enzyme (Dr. Robertson). 

In collaboration with New England Nuclear 
Corp. L-proline-3,4-H 3 has been made available 
to biochemists for metabolic studies. There has 
always been a great need for such a selectively 
tritiated optically pure proline (Dr. Robertson). 

In collaboration with the Laboratory of Clini- 
cal Biochemistry and Dr. E. Katz, Special Fellow 
from Rutgers Univ., the modification of actino- 
mycin through addition of dehydroproline and 
many of its derivatives to the culture medium of 
Streptomyces griseus is being investigated. The 
aim in this study is the elaboration of less toxic 
and more carcinostatic actinomycins and to gain 
more information on the enzymes that incorpo- 
rate such foreign amino acids as pipecolic acid, 
azetidinecarboxylic acid, etc. into the peptide 
part of actinomycin (Dr. Robertson). 

In collaboration with Dr. A. Berger, The 
Weizmann Inst, of Science, Rehovoth, Israel, 
poly-3£-dehydro-L-proline has been made by 
polymerization of N-carboxy-3,4-dehydro-L-pro- 
line anhydride. The residue rotation and muta- 
rotation of this polymer was determined and 
compared with those of poly-L-proline, which is 
assumed to have a righ-handed helical configura- 
tion in solution (Dr. Robertson). 

A simple qualitative visual test for the detec- 
tion of monamine oxidase in tissue slices has 
been developed; it is based on the slow conver- 
sion of dehydronorkynuramine to indigo by oxi- 
dative deamination and intramolecular cycliza- 
tion to indoxyl which is rapidly autoxidizeed to 
indigo, whose substantivity without addition of 
further aromatic substituents is, however, not 
sufficient for a useful histochemical staining 
method (Dr. Y. Kanaoka). 

In cooperation with Dr. Axelrod, NIMH, 
studies on catechol-0 -methyltransj 'erase and on 
the equilibrium with the products arising from 
the action of microsomal O-demethylase have 
been carried into the mescaline series utilizing 
as substrates mescaline, mescalol and mescalone 
on the one hand, and derivatives of 3,4,5-trihy- 



droxyphenethylamine on the other hand. So far 
4 partly phenolic metabolites of mescaline have 
been observed and identified by synthesis. The 
pharmacological aspects of these metabolites are 
being worked out in cooperation with the Ster- 
ling- Winthrop Research Institute (Dr. J. Daly). 
In collaboration with the Laboratory of Clini- 
cal Biochemistry, the mechanism of the action of 
monamine oxidase is being investigated. It has 
been found in this cooperative investigation that 
dimethyltryptamine N-oxide is degraded by mon- 
amine oxidase at a rate significantly depending 
on the partial pressure of oxygen present (Dr. 
Y. Kanaoka) . 

In collaboration with Regis Chemical Com- 
pany, Chicago, and aided by a special grant from 
Psychopharmacology, kynuramine dihydrobro- 
mide has been made available to clinical and 
biochemical laboratories as the standard sub- 
strate of choice for the rapid and routine spec- 
trophotometric assay of monamine oxidase. By 
the same arrangement homocarnosine, the new 
peptide of v-aminobutyric acid with L-histidine, 
which has been isolated from brain tissue in the 
Laboratory of Clinical Biochemistry, has been 
made available in research quantities. Similarly, 
serotonin as the crystalline acid oxalate, has for 
the first time been made available to biochemists 
and clinicians with none of the drawbacks that 
the previous serotonin-creatinine complex had. 

In collaboration with Dr. S. L. Friess and 
Chief Durant of the Naval Medical Center the 
labilization of ester bonds in aminocyclitol de- 
rivatives has been further investigated. The 
symmetric and asymmetric di- and tri-O-acetates 
of N,N'-tetramethyl-2-deoxystreptamine have 
been synthesized, purified and subjected to 
kinetic analysis of their hydroylsis rates. A 
combination of field, charge-transfer and over- 
all conformational effects combine to impart to 
some of these esters a liability which, with a 
half-life time of 19 minutes, surpasses that of 
p-nitrophenyl acetate by more than 100 times. 
These models deserve consideration in connec- 
tion with model substrates for cholinesterase 
(Drs. Kny and Daly) . 

Cooperative studies with the Laboratory of 
Clinical Biochemistry, NHI, on the mechanism 
of hydroxylation of dopamine to norepinephrine, 
and of tyramine to octopamine utilizing selec- 
tively tritiated precursors have yielded evidence 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



for the existence of an enzyme-substrate inter- 
mediate in which the tritium atoms at the 
methylene group next to the aromatic nucleus 
exchange with the solvent. The elaboration of 
this phenomenon into a quantitative assay for 
dopamine-/J-oxidase (norepinephrine synthetase) 
is in progress (Dr. Y. Kanaoka). 

In collaboration with the Laboratory of Clini- 
cal Biochemistry procedures have been developed 
for the detection of inhibitors of monamine oxi- 
dase in vitro and in vivo and for the determina- 
tion of the duration of their central and periph- 
eral action. These procedures have been applied 
to the detection and study of several new classes 
of inhibitors (Dr. M. Ozaki). 

It has been demonstrated in several studies 
that the presence of tertiary butyl groups ortho 
to a phenol greatly increases the contribution of 
dienone structures to the resonance hybrid. The 
effect of such a difference on spectral and ioniza- 
tion properties has been reported as well as 
changes in the rate of several reactions. Stable 
phenolic hypobromites have been detected and 
methods have been elucidated for stabilizing free 
radical intermediates analogous to those occur- 
ring in the biosynthesis of thyroxine (Dr. L. A. 
Cohen and Mr. W. B. Jones). 

Steroids 

Anthrasteroid rearrangement: The two iso- 
meric anthrasteroid alcohols (acid catalyst, p- 
toluenesulfonic acid*H 2 0) have the secondary 
hydroxyl in ring A (either position 2 or 3) ; 
dehydroxylation leads to hydrocarbons isomeric 
at Ci 4 ; the mechanism accommodating the for- 
mation of these isomers must be complex and is 
at present completely obscure. By rearrange- 
ment of 3-desoxydehydroergosterol, depending on 
the conditions, either the 14a- or the 14/?-anthra- 
ergostatriene is formed. Finally and most sur- 
prisingly, the i^a-anthraergostatrienol acetate (as 
only product) was obtained by boiling dehydro- 
ergosterol in acetic acid (Dr. O. Tanaka and 
Mr. J. Steele). 

Thiol analogs of corticoids : A new method for 
converting thiocyanoderivatives to thiols via the 
acetyldithiocarbamates has been developed and 
applied to 3a,9a-oxido-ll/?-thiocyano-5/?-andro- 
stan-3/?-ol-l7-one-3-methylether and the corre- 
sponding corticoid (Dr. Y. Ueda). 

Stevioside: The only uncertainty left in the 



structure of stevioside is the position of the 
carboxylic group, i.e., whether at C* or Cio. Ex- 
periments are being conducted to degrade the two 
di hydrosteviols to the corresponding 13-desoxy 
primary alcohols and to compare these directly 
with the analogous degradation products from 
Djerassi's cuauchicicine (Dr. U. Beglinger). 

Steroidal Alkaloids: The four possible C22 5 
C 2 5-solanidan-3-ones have been prepared. A new 
method with commercial potential has been de- 
veloped for the conversion of solasodine to preg- 
nadienolone via the pyridine hydrochloride isom- 
erization. Solasodine and tomatidine have been 
converted via the "oximino ketones" to the re- 
spective bisnorcholenic and bisnorcholanic acid 
16 — ->- 22 lactones, of possible value as aldos- 
terone antagonists (Drs. Sato and Erekawa). 

Steroid analysis : A method has been developed 
for rapid and accurate plating of radioactive 
steroid fractions. A fully automatic steroid 
analyzer has been constructed and is undergoing 
final testing. Major points: Preadjustment for 
any desired ratio of elution solvents; automatic 
determination of the adrenal corticoids by U.V. 
absorption and reducing power; recording of re- 
sults on a strip chart (Drs. Johnson and Heft- 
mann) . 

Adrenal cortical hormones in rat adrenal tu- 
mor tissue: While in the normal rat adrenals 
corticosterone and aldosterone are the end prod- 
ucts of metabolism, tumor tissue showed a greatly 
decreased ability for 11-hydroxylation of added 
precursors (e.g. progesterone). This, in turn, 
leads to an accumulation of 11-deoxycorticos- 
terone. The latter has not been isolated from 
rat adrenals heretofore (Drs. Johnson and Heft- 
mann). 

Steroid analysis by gas chromatography : The 
steroidal alkaloids solasodine and tomatidine and 
a number of their derivatives have been chroma- 
tographed in microgram quantities, on a column 
of Chromosorb W, 80-100 mesh containing 0.75 
percent SE-30. The response to the finest steric 
differences makes possible the detection of a 
number of stereoisomers in a complex mixture 
(Drs. Sato and Ikekawa). 

IR spectroscopic studies: Additional spectra 
have been converted to proper units for Lorentz- 
ian analysis. A Rudolph Recording Speetro- 
polarimeter acquired recently has been put in 
operating condition to produce usable optical 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



169 



rotatory dispersion data. Both Model-21 spec- 
trophotometers have been equipped with auxiliary 
recorders for presentation of spectra on N.B.S. 
punch cards (Mr. H. K. Miller and Mrs. A. W. 
Wright). 

Biogenesis of plant sterols: "C 2 -acetate or 
w C2-mevalonate were injected into tomato fruits. 
Stigmasterol was isolated (A 5,22 -stigmasta-dien- 
3/?-ol) in pure form (and its radioactivity deter- 
mined) together with some other active appar- 
ently sterol-like components. The slime mold 
Dictyostelium discoideum has been grown on a 
medium containing 14 C-mevalonate or "C 2 ace- 
tate. The activity of stigmastenol (A^-stigmas- 
ten-3/3-ol), isolated in pure form, was determined 
(Drs. Heftmann, Bennett, and Johnson). 

Biogenesis of steroid sapogenins: Homogen- 
ized tubers of Dioscorea floribunda were incu- 
bated with radioactive acetate or mevalonate. 
While the acetate was incorporated in a low but 
constant rate into diosgenin, no trace of activity 
could be detected in the diosgenin from the 
mevalonic acid incubates. Several other radio- 
active products have been isolated but not yet 
structurally elucidated. There is an appreciable 
difference of biosynthetic activity in the various 
parts of the tuber; the highest synthetic rate is 
found in the portion around the tuber (Drs. 
Heftmann and Bennett). 

Cooperative work: In cooperation with the 
Section on Physiology, LPD, MAID (Dr. von 
Brand) , the Insect Physiology Lab., ARS, Dept. 
of Agric. (Dr. S. J. Louloudes), and the Section 
on Metabolism, LCPM, NHI (Dr. Y. Avigan), 
complete analysis and characterization of a num- 
ber of steroids (sterols) from the extract of 
helminths, and insects (reared under artificial 
condition) have been carried out. Desmosterol 
has been isolated (rat liver) and characterized 
in the "cholesterol-biosynthesis" inhibited by 
MER-29. 14 C-labeled desmosterol, -methostenol 
and -lanosterol have been and are being synthe- 
sized in connection with the above biosynthetic 
work. 

Analytical Services 

Approximately 10,000 determinations were 
made of which two-thirds were for carbon, hy- 
drogen and nitrogen, the remaining third for 
halides, sulfur, phosphorus, functional groups, 
selenium, various metals, optical rotations, weight 



losses, etc. These services were utilized by ap- 
proximately 130 research scientists at the NIH. 
A limited number of analyses were performed 
for other government agencies. Elemental analy- 
sis for fluorine was made available during the 
year. A study is being made of the direct deter- 
mination of oxygen in organic compounds and 
it is hoped that this analysis will be available 
by the first part of next year (Mr. McCann, 
Miss Parisius, Mrs. Peake, Mr. Baer, Mrs. Wong) . 

Laboratory of Pathology & Histochemistry 

Altitude Effects 

Drs. Benjamin Highman and Paul D. Altland 
found that dogs exposed to simulated high alti- 
tude show a transient hyperglycemia, and a sharp 
rise (lasting 3-7 days) in serum glutamic-oxal- 
acetic transaminase (SGO-T), serum glutamic- 
pyruvic transaminse (SGP-T), serum lactic 
dehydrogenase (SLD) and serum alkaline phos- 
phatase (SAk-P). The rise in SGO-T, SGP-T 
and SLD is diminished by the adrenergic block- 
ing agent phenoxybenzamine and the hyper- 
glycemia by the ganglionic blocking agent 
chlorisondamine. These findings support the 
view that altitude hypoxia produces hypergly- 
cemia by release of endogenous catechol amines 
and elevates serum enzymes by altering cellular 
permeability. Drs. Altland and Highman have 
found that cholesterol-fed rabbits, exposed to 
16,000 feet, up to 17 weeks, show much more 
severe pulmonary atheroclerosis, less severe le- 
sions in the descending and abdominal aorta, and 
a much higher incidence of marked calcium de- 
posits in both aortic and pulmonary athero- 
sclerotic lesions; hypoxia and pulmonary hyper- 
tension are considered major contributing factors. 
Drs. F. Smith, Altland, and Highman find that 
rats acclimatized to altitude and injected with 
sheep erythrocytes show significantly increased 
(over ground level) antisheep erythrocyte hemo- 
lysin, formed during the primary immune re- 
sponse. 

Cytogenetic Studies 

Dr. J. H. Tijo in continuing collaborative study 
with several investigators on the chromosomal 
complement of patients with leukemia and with 
congenital anomalies, i.e. ovarian dysgenesis, 



170 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Turner's syndrome has revealed karyotype ab- 
normalities in a number of such patients. 

Collaboration with NCI an investigation of 
the chromosomal constitution of a series of trans- 
plantable thyroid tumor lines, in an attempt to 
relate properties of these lines with their chro- 
mosomal pattern, has revealed a constant chro- 
mosomal defect in the stem line of two non- 
functional anaplastic lines and on the stem line 
of an only moderately de-differentiated line. 
Also in collaboration with NCI, Dr. Tijo has 
developed a simplified method for the study of 
chromosomes of bone marrow cells without prior 
in vitro growth which has been used with good 
results on rat, mouse, hamster and man. 

Degenerative Joint Disease and 
Human Rheumatism 

Dr. Leon Sokoloff extended studies of the 
pathogenesis of degenerative joint disease and 
of the descriptive pathology of human rheuma- 
tism. A comprehensive analysis of the genetics 
of osteoarthritis in mice is nearing completion. 
The joint disease is transmitted as a recessive 
characteristic; more than one gene appears to be 
involved ; there is no sex linkage. Studies, made 
in collaboration with Dr. R. S. Yamamoto, dis- 
close that there are significant genetic differences 
in plasma lipids, hepatoma-formation and sev- 
eral other lesions in the same animals, but that 
there is no correlation between these findings and 
the joint disease. Comparative pathology of de- 
generative joint disease continues to be investi- 
gated. A study on vertebral coalescence in the 
giant dinosaur Diplodocus longus has been com- 
pleted in collaboration with Dr. B. S. Blumberg. 
This is surmised to represent a traumatic osteo- 
arthritis. 

A paper on the pathogenesis of ochronotic 
arthropathy, written with Drs. William O'Brien 
and W. G. Banfield, has been accepted for pub- 
lication. The data suggests that the critical 
problem in the development of the joint disease, 
as distinguished from the biochemical defect, is 
affinity of articular tissues for the homogentisic 
acid or its pigment derivatives. 

Germ-Free Animals — Nutritional Deficiencies 

Using germ-free mice Dr. David Beaver found 
that injected progesterone was quite capable of 
producing the typical vaginal neutrophilic exu- 



date seen in certain phases of the estrous cycle; 
bacterial infection is not required. 

In another study by Dr. David Beaver, the 
A-deficient, germ-free rat, similar to the conven- 
tional animal, exhibits widespread focal keratin- 
izing metaplasia of mucous membranes. Germ- 
free animals, in addition, show degeneration or 
necrosis of the liver, kidney, heart, and adrenal 
gland. The germ-free state and the absence of 
infection modify the pathogenesis and histologi- 
cal appearance of the lesions of A deficiency, but 
do not prolong life. The latter fact suggests 
that in Vitamin A deficiency there is a significant 
metabolic disturbance. 

Dr. L. L. Ashburn in cooperation with the 
Walter Reed Army Institute of Research, has 
shown that a low protein, choline deficient diet 
leads to liver cirrhosis in germ-free rats. In 
fact, the development of the lesion appears to 
progress faster than in conventional animals. 
These findings are being further studied as is the 
mechanism whereby certain antibiotics appear to 
delay onset and progress of the cirrhosis. Drs. 
Beaver and Ashburn have found that the lungs 
of these rats and those of germ-free rats on a 
pantothenic acid deficient diet show prominent 
lipid deposits visible on gross examination as few 
to abundant white spots of 1 to 3 mm size. These 
nodules are formed of xanthoma cells containing 
triglycerides and lesser amounts of anisotropic 
material. Although the lesions are seen occa- 
sionally in other circumstances, germ-free state 
and the specific abnormal diet are necessary for 
its maximum expression. 

Goldthioglucose Obesity 

Radioautography of tissues submitted to acti- 
vation analysis permits localization of gold in 
mice made obese by goldthioglucose. This study 
by Dr. George Brecher shows that areas of acute 
necrosis seen at 12 hours correspond to gold 
localization. Later, minute scars contain the 
bulk of the gold. In other experiments it was 
shown that glucose cannot effectively compete 
with goldthioglucose in localization in the hy- 
pothalamic feeding center, as is implied in 
Mayer's widely accepted glucostatic theory. 

Hemoglobin 

The study of hemoglobin by Dr. Harvey Itano 
and Miss Elizabeth Robinson has continued to 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



171 



furnish new information on the structure, genetic 
control, biosynthesis, and evolution of a protein 
molecule. The ability of the subunits of canine 
hemoglobin to combine with the subunits of both 
normal and abnormal human hemoglobins sug- 
gests retention of the characteristic tertiary struc- 
ture in spite of changes in primary structure. 

! Studies on the inheritance of the two types of 

: polypeptide chains of hemoglobin have yielded 
the first example of a protein synthesized under 
the combined control of two nonadjacent genetic 

'■ loci. The chains are apparently synthesized in- 
dependently in identical pairs and a doubly 
heterozygous red cell synthesizes four different 
chain-pairs which combine to form four different 

; hemoglobins. 

In continued studies of abnormal human hemo- 
globin-! (Hb-I) , Dr. Makio Murayama has found 
that the genetic alteration takes place in the 
alpha chain (N-terminal val. leu....) of the mole- 
cule. In contrast, the chemical abnormalities of 
Ilb-S, Hb-C, and Hb-G are known to be located 
in the beta chain (N-terminal val.his.leu....). The 
amino acid sequence studies revealed the fol- 
lowing : 

Hb-A val.leu....ala.val.try.gly.lys.... 
Hb-I val.leu....ala.val.try.gly.asp.... 

A lysine residue is replaced by aspartic acid in 
this genetic alteration. 

Hematology 

The kinetics of red cell proliferation have been 
further elucidated by Dr. Frederick Stohlman. 
Erythropoietine has been shown to act by pro- 
moting the differentiation of primitive stem cells 
into erythroid elements; some of these cells ma- 
ture without dividing ; a substantial number when 
severe hypoxia exists. Erythropoietine, appar- 
ently, does not directly affect the erythroid ele- 
ments (i.e. pronormoblasts, etc.). Preliminary 
evidence suggests that these rapidly maturing 
cells have a shortened life span. 

The effectiveness of erythropoietine in the ir- 
radiated animal has been explained by studies 
in which it was shown that depopulating the 
stem cells compartment by administering ery- 
thropoietine immediately after irradiation serves 
as a stimulus for earlier regeneration of the stem 
cell compartment. This implies that one mech- 
anism for regulation of the stem cell compartment 



is the total number of cells, whether fertile (i.e. 
capable of division) or infertile, and that differ- 
entiation of stem cells into erythroid elements 
results in an increased rate of division amongst 
the remaining stem cells. 

Histochemistry 

Studies on the localization in tissue sites of 
polysaccharide synthesis by Dr. Tadao Takeuchi 
have resulted in the development of a histochemi- 
cal method for the cellular localization of enzy- 
mic activity forming polysaccharides from uri- 
dinediphosphate glucose (UDPG) . This method 
localizes UDPG glycogen transferase activity to 
numerous tissue sites and its activity has been 
compared to that of amylophosphorylase in these 
areas. Further investigation has resulted in the 
development of a method for the histochemical 
localization of uridinetriphosphate-pyrophos- 
phorylase activity in tissue sections. 

A continuation of studies by Dr. George Glen- 
ner on the localization of proteolytic enzyme ac- 
tivity in pathologic and normal tissue has been 
extended, using newly synthesized chromogenic 
substrates to the demonstration of an esterolytic 
enzyme having several characteristics distinct 
from those previously described histochemically 
and to the investigation of an enzyme hydro- 
lyzing a specific class of amide substrates. 

In order more accurately to demonstrate the 
sites of numerous dehydrogenases in tissue sec- 
tion with the elimination of false positive locali- 
zation a new route for the synthesis of pure 
ditetrazolium salts has been developed. A screen- 
ing method for the demonstration of Endamoeoae 
based on their glycogen content (a modified 
methenamine silver technique) has been applied 
to pathologic tissues for routine diagnostic pur- 
poses. 

Dr. Samuel Spicer has partially characterized 
mucins in normal rodent and human tissues and 
in human pathological material by correlative 
studies with established autoradiographic and 
histochemical staining methods and by newly 
developed procedures. The methods developed 
for histochemical identification of mucins include 
combinations of conventional staining techniques, 
a periodic acid diamine procedure, and enzymatic 
digestion with sialidase. Application of all the 
available specific techniques reveals varieties of 
sulfomucins and sialomucins not known to exist 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



from biochemical methods. Such studies also 
suggest the possibility that the histochemical 
properties of the mucins in normal as well as 
pathological tissues may be unique in each histo- 
logic site. In other studies, cytosiderin and 
peracetic acid-aldehyde fuchsin stained (lipofus- 
cin?) bodies observed in epithelial cells in a 
number of organs appear to be, in some instances 
at least, associated with strong acid phosphatase 
activity. The cytoplasmic particles are at pres- 
ent considered to be a possible morphologic mani- 
festation of the catabolic removal of discarded 
cytoplasmic macromolecules. 

Dr. David Beaver, in an extensive investiga- 
tion of the rat preputial gland found that the 
structure varies greatly with the fixative used. 
In addition to lipids, the acinar cells contain 
numerous protein granules. A stain was devised 
which permitted the demonstration of both ele- 
ments in the same section. With this and other 
histochemical methods the appearance (function) 
of the gland was evaluated in relation to sex, age, 
androgenic and estrogenic stimulation, and hypo- 
physectomy. 

Human Pathology 

The opportunities offered to this laboratory 
through consultative and diagnostic studies of 
surgical and autopsy specimens from the Indian 
Health Program of the Public Health Service 
have continued to stimulate interest in aspects 
of geographic and environmental pathology. Di- 
rect contacts with these Hospitals during the 
year promises to improve greatly the quality of 
the material for study. Problems related to sar- 
coidosis, to dietary hemosiderosis, to diabetes, 
and to atherosclerosis and its complications 
among the Indians are of particular interest. 

Attempts by Drs. Gert Laqueur and Harry 
Marsh to isolate in culture the organism respon- 
sible for Pneumocystis carinii pneumonitis from 
animal and man have been continued throughout 
the year. All experiments failed, using culture 
media for fungi and protozoa and specialized 
bacteriologic media under aerobic and anaerobic 
conditions with varying acidity, viscosity and 
nutrients. However, recent attempts, with the 
collaboration of Mr. Charles Zierdt of the micro- 
biology division of the Clinical Pathology Labo- 
ratory, have produced the first promising results 
utilizing highly specialized procedures. It has 



been possible to obtain the same organism from 
several animals in culture. The organisms tinc- 
torially and morphologically resemble closely 
those seen in the human and animal disease; 
introduction into germ-free animals is expected 
to establish their pathogenicity. 

Immunology 

Dr. Emily Emmart, using fluorescent antibody 
and other techniques has shown that: (1) strep- 
tococcal hyaluronidase is localized in certain 
tissues of infected mice after in vivo elaboration 
of the enzyme; (2) glyceraldehyde-3-phosphate 
dehydrogenase is present in the I-band (mito- 
chondria) of the wing and leg muscle of the 
roach (Periplaneta americana) ; (3) myosin is 
localized in the myofibrils (A-band) of the con- 
duction fibers of the beef heart. 

In collaboration with Dr. Marion Webster im- 
munochemical studies on the antibodies to human 
kallikrein preparations in successive stages of 
purification have been carried out. The rise in 
gamma globulin has been followed by paper 
electrophoresis on sera of immunized rabbits. 
Antibody has been determined by precipitin re- 
actions in vitro, in agar and by antibody inhibi- 
tion of the vasodilator action of kallikrein in the 
dog. 

Dr. Edwin Lerner, with the collaboration with 
Drs. Kurt Bloch and J. J. Bunim has shown 
that rabbits immunized repeatedly with their own 
fractionated gamma globulins failed to show 
positive reactions for rheumatoid factor, or for 
anti-human antibody. This is in contradistinc- 
tion to the results of Milgrom, Witebsky, et al, 
and may conceivably be due to the rigorous pre- 
cautions taken to exclude contamination by hu- 
man, other animal, or other rabbit proteins. Drs. 
McMaster, Lerner and Exum have shown that ex- 
perimental allergic thyroiditis has been produced 
in inbred guinea pigs and has been shown to 
correlate with the presence of delayed hypersen- 
sitivity, but not with the presence of circulating 
anti-thyroid antibody. The allergic thyroiditis 
was the earliest ever produced experimentally, 
and the most severe at the time intervals studied. 
The experimental conditions excluded direct 
trauma to the thyroid, sensitized reaction to 
thyroid, and immunologic interference by hetero- 
logous antigenic serum or tissue proteins. 



NATIONAL INSTITUTE OF ARTHKITIS AND METABOLIC DISEASES 



173 



Renal Structure and Function 

The functions of the straight and convoluted 
segments of the rat proximal tubule which differ 
distinctly with respect to their handling of 
phenol or clorphenol red, were investigated by 
Dr. James Longley in respect to a number of 
other phenolsulfonephthalein dyes. Several dyes 
were found to behave similarly to the ones previ- 
ously studied. Others were found to be accumu- 
lated by the convoluted segment only and still 
others to show no apparent accumulation at all. 
No correlation between the physical or chemical 
characterisitcs of the dyes studied and their han- 
dling by the kidney has yet been established. 

In addition to the research projects abstracted 
above, certain scientists in other laboratories re- 
ceive advice from members of our staff; particu- 
larly in pathologic anatomy. Our histopatho- 
logical preparation unit also took part in this 
cooperative effort by cutting and staining about 
2,000 tissue sections this year for ten investiga- 
tors not in laboratories of NIAMD. 

Laboratory of Pharmacology and Toxicology 

Spermidine and Spermine 

A striking development in the studies on the 
polyamines has been the observation that these 
amines, even in very low concentrations, have a 
marked stabilizing effect on protoplasts, mito- 
chondria and bacteriophage T5. The mechanism 
of this effect is not clear, but may be related to 
the binding of these amines to nucleic acids and 
phospholipids. 

Further work has also been carried on the en- 
zymes and intermediates concerned in the bio- 
synthesis of spermidine. Thiomethyladenosine 
has been isolated and identified as the product 
of the reactions: Putrescine + decarboxylated 
adenosylmethionine ->- spermidine + thiomethyl- 
adenosine. 

Under certain conditions much of the spermi- 
dine of E. coli is present as a glutathione-con- 
taining derivative. A tentative structure has 
been presented for this derivative (Drs. H. Ta- 
bor, C. W. Tabor, S. M. Eosenthal, G. Jamieson 
and T. Viswanatha). 

Histidine and Related Compounds 

1. The reduction of unsubstituted imidazole 
and its derivatives has been achieved for the first 



time. This has been accomplished by carrying 
out the catalytic hydrogenation of these com- 
pounds in acetic anhydride. Furthermore, by 
this method new imidazoline and imidazolidine 
compounds have been prepared. 

2. Imidazoleacetic acid ribotide is formed by 
the following enzymatic reaction: Imidazoleacetic 
acid + pyrophosphorylribosephosphate (PPRP) 
+ ATP -> imidazoleacetic acid ribotide + ADP 
+ orthophosphate + pyrophosphate. 

Hydrolysis of imidazoleacetic acid ribotide re- 
sults in imidazoleacetic acid riboside, which we 
have previously shown to be present in the urine 
after histamine and imidazoleacetic acid admin- 
istration. 

3. The reaction histidine ->- urocanic acid + 
NH 3 proceeds via an ammonia-enzyme inter- 
mediate. 

4. The following reaction has been demon- 
strated in an enzyme purified from E. coli: 
Ergothioneine ->■ thiourocanic acid + trimethyl- 
amine (Drs. H. Tabor, G. Crowley, J. Wolff, H. 
Bauer, A. Peterkofsky and Miss V. Childs). 

Sialic Acid. 

1. A hitherto undescribed enzymatic system 
has been purified from mammalian liver for the 
biosynthesis of N-acetylneuraminic acid. 

2. A sialidase has been purified from a bovine 
serum fraction (VI). This is the first time a 
sialidase has been described in mammalian tis- 
sues. 

3. The enzymatic removal of sialic acid from 
different genetic forms of human transferrin has 
revealed that part of the structure of the differ- 
ent forms is common to all. Sialic acid is prob- 
ably responsible for the differentiation between 
the various genetic forms of part of the trans- 
ferrin molecule (with Dr. Blumbery). 

4. Other studies have included the determina- 
tion of the concentration of sialic acid in cere- 
brospinal fluids (with Dr. R. Jakoby) ; the dem- 
onstration that one-third of the sialic acid of 
cerebrospinal fluid is bound to small sized di- 
alyzable material; a test for sialic acid-contain- 
ing mucins in tissue and in tumors using a histo- 
chemical procedure (with Dr. Spicer) ; a study of 
the amount and nature of the sialic acid in a 
variety of nonmammalian organisms; and the 
demonstrations of sialic acid in an ether-soluble 



174 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



lipid in sea urchin eggs (Drs. L. Warren, H. 
Felsenfeld, and Miss C. Spearing). 

Burns and Shock 

Studies have been continued on the therapy 
of burns in clinical cases in Peru. In a large 
series of adult cases oral saline was as effective 
as a combination of saline and plasma therapy. 

The occurrence of infection in burn cases con- 
tinues to be a serious problem at all ages; chil- 
dren under 3 years were particularly susceptible. 
The incidence of septicemias in these children 
could be decreased by large doses of plasma or 
gamma globulin, with some lowering of the mor- 
tality. Antibiotic therapy, on the other hand, 
had no effect. 

In the laboratory a bacteriological study of 
burned mice (with Dr. E. Verder, NIAID) sug- 
gested that after burn injury, bacteria of low 
virulence and invasiveness are able to spread 
from the local area and establish generalized in- 
fections (Drs. S. M. Rosenthal, R. C. Millican, 
N. A. Kefalides, K. Markley, and Peruvian as- 
sociates). 

Mouse Leprosy 

In the field of experimental leprosy better 
conditions for the culture of macrophages have 
been developed. Macrophages can now be main- 
tained up to 100 days, permitting cultivation of 
Mycobacterium leprae murium. This represents 
a marked improvement over the previously ex- 
isting techniques which only permitted survival 
of macrophages for a short time (usually less 
than 10 days). This is of considerable import- 
ance for leprosy studies, as this has permitted 
survival and growth of the Mycobacterium leprae 
murium in a given tissue culture for long periods 
(Dr. Y. T. Chang). 

Sulfur Amino Acids 

Three enzymes have been isolated from yeast 
that are required for the reaction: 

+ 
L(-) Methionine sulfoxide + TPNH +H -> L- 

methionine + TPN + H 2 0. 

In addition to this reaction two of these en- 
zymes carry out the TPNH-catalyzed reduction 
of dithiols to thiols (Drs. S. Black, J. F. Thomp- 
son, and Miss Hudson). 



Enzyme Activity 

1. The various enzymes of the urea cycle in- 
crease with an increase in dietary protein, and 
decrease with a lowered dietary protein (Dr. 
R. T. Schimke). 

2. Trypsin and chymotrypsin have been shown 
to contain hydroxylysine. This amino acid has 
not been described previously in an enzyme pro- 
tein (Dr. Viswanatha with Dr. Irreverre). 

3. The ability of chymotrypsin to react with 
diisopropylfluoridate or p-nitrophenylacetate was 
separated from its ability to hydrolyze peptide 
bonds by the differential destruction of the latter 
activity by treatment with N-bromosuccinimide 
(Dr. Viswanatha with Dr. Lawson). 

4. Spectrophotometric methods have been de- 
veloped for the measurement of the aldolase-di- 
hydroxyacetone phosphate complex for kinetic 
studies on this enzyme after alteration by car- 
boxypeptidase (Dr. Mehler and Miss Asnien). 

Excitable Cells 

Further evidence has been obtained that cal- 
cium transfer is at least one direct factor in the 
coupling between excitation and muscular con- 
traction in the fibers of the "twitch," "slow" 
and cardiac type. Additional correlations have 
been established between the interactions of vera- 
trum alkaloids with monomolecular films of 
stearic acid at air-water interfaces and with liv- 
ing membranes (Drs. A. M. Shanes, N. L. Gersh- 
feld, S. Winegrad, S. Dikstein, and C. P. Bian- 
chi). 

Gramicidin J 

During studies on the enzymatic biosynthesis 
of gramicidin J in extracts of Bacillus brevis 
the enzymatic formation of a dipeptide of 
phenylalanine and proline was observed, which 
may be an intermediate in gramicidin biosyn- 
thesis (Dr. K. Kurahashi). 

Cholesterol Synthesis 

A rabbit liver enzyme has been purified which 
converts phosphomevalonic acid to pyrophos- 
phorylmevalonic acid (Dr. K. Markley and Mrs. 
Smallman). 

Office of Mathematical Research 

Work has continued along the major lines in- 
dicated in last year's report, viz. : Mathematical 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



175 



formulation and analysis of neurophysiological 
problems (Dr. Wilfrid Kail, Mr. Ezra Shahn 
and Mrs. Jeanne Altmann) ; development of 
mathematical and computational methodology for 
assessing mathematical models based on differen- 

i tial equations and detailed studies of iodine me- 
tabolism in the thyroid system (Dr. Mones Ber- 
man, Mrs. Marjorie Weiss, Mr. Ezra Shahn and 
collaborators from other groups as noted below) ; 
general mathematical problems arising from the 

i rate of behavior of biological systems (Dr. John 

• Hearon) . Work has been initiated in visual per- 
ception and pattern recognition (Mrs. Rosalind 

i Marimont, NIMH, Associate Member of OME). 
The work along these four lines is summarized, 
in the same order, below. It is not possible to 
give all of the results of the year's work and in 

: each case only highlights and/or examples are 
included. 

The solution for the external and internal field 
for a neuron with spherical soma and cylindrical 
dendrites has been obtained. Such fields have 
been computed numerically on the IBM-650 for 
a neuron with seven asymmetrically placed den- 
drites. In particular, fields were computed for 
the instants in time when the "A-spike" and "B- 
spike" reach their maximum potential. These 
detailed numerical calculations have yielded re- 
sults of broad qualitative significance for the in- 
terpretation of neurophysiological recordings. 
The mathematical treatment of the spread of 
electric current between soma and branching den- 
dritic trees has been extended to the transient 
case. This information allows interpretation and 
analysis of experiments designed to estimate the 
membrane time constant and elucidate the nature 
of synaptic potential. 

The program, written for the IBM-704, orig- 
inally developed for analysis of tracer kinetics 
has undergone major revision and consolidation 
and major extension in terms of generality and 
flexibility. In particular the program will ac- 
cept input data directly in a variety of forms, 
take account of dependence relations among two 
or more parameters, take quantitative account of 
the information available on a given compart- 
ment or sub-system in fitting a particular com- 
partment. The program has been adapted to 
non-linear systems and as a result can be made 
to perform the analysis of any mathematical 
model based on an arbitrary set of differential 



equations. The general program is available to 
any investigator. The program, and the mathe- 
matical methods which it was designed to imple- 
ment, has had considerable application: Labeled 
glucose studies in collaboration with Dr. S. Segal, 
NIAMD, thyroxine studies in collaboration with 
Dr. H. Haddad, Washington University; a vari- 
ety of problems in collaboration with Drs. S. 
Wollman and M. Elkind, NCI, and Drs. D. Stein- 
berg and J. Davies, NHI. 

It has been shown that if A is a matrix of 
order n with non-positive diagonal and non- 
negative off-diagonal elements then no component 
of a vector x, initially nonnegative and obeying 
dx/dt = Ax, can vanish at a finite time. The 
original intent was to demonstrate that a rate 
matrix with the above sign pattern guarantees 
positive concentrations, and conversely positive 
concentrations imply that sign pattern. The 
mathematic statement of this commonsense con- 
straint on a real system leads to the following 
practical results : It follows that a necessary con- 
dition for overshoot in a linear system is that 
some initial concentration be below and some 
above the steady-state value; that, if EjAij < O, 
every element of -A" 1 is non-negative, the small- 
est root is simple, and the co-factors of A have 
the sign of (-l) n+1 . It has been shown that a 
function F(x,y) symmetric and homogeneous of 
degree zero has the property aF/ax = aF/ay 
= O on the line y = x. This theorem, necessary 
in the error analysis of certain approximate dif- 
ferential equations for metabolic systems, has the 
consequence that class of function having an 
optimum which is a point of symmetry in the 
logarithmic plot can be defined and the class of 
mechanism which may underlie optimum pH, 
substrate-concentration, etc., can be stated. 

Work has recently been initiated on a mathe- 
matical study of visual perception with special 
emphasis at present on color vision. The main 
objective is a mathematical treatment of the em- 
pirical laws of color vision designed to produce 
the necessary and sufficient conditions required 
by them. It has been shown that Grassman's 
laws and Abney's law are expressions of a mathe- 
matical property which has been denoted as 
quasi-additivity. In terms of functions : if f (x) 
= f(y) and f(u) = f(v) then f(x+u) = f 
(y+v). In terms of the laws of color vision f 
will be some general transformation and x, y, u, 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



v may be spectral distributions or functionals of 
them and equality is then defined as "matching" 
of lights. It has been shown that if f is a one- 
to-one function of a linear operation Abney's 
law is implied. The converse of this is being 
studied in terms of more general spaces and the 
similar but more general problem of Grassman's 
laws is being pursued. 



CLINICAL INVESTIGATIONS 

The clinical and laboratory research activities 
of the Clinical Investigations area of N.I.A.M.D. 
have continued to increase during the past year. 
No new units have been added nor additional 
space acquired. 

The complement of patients beds has been ex- 
panded from 65 to 70 as of March 1, 1960 and 
the census for the year has averaged 76 percent. 

A totalof 483 in-patients was admitted during 
the 12-month period from December 1, 1959 to 
November 30,, 1960, an increase of 94 patients 
(24 percent) over the same period last year. 
The total patient days was 19,134, an increase of 
2,192 over the preceding year. In the Admissions 
and Followup Department 1,585 patients were ex- 
amined and studied, an increase of 65 over the 
past year. The average in-patient stay at the 
Clinical Center was 40 days. 

Investigations related to the diseases studied at 
N.I.A.M.D. have resulted in 94 publications in 
scientific journals, monographs, annual reviews 
and medical textbooks. During the past year 
Dr. Joseph J. Bunim was awarded the Heberden 
Medal by the Heberden Society of Great Britain 
in recognition of his research in the rheumatic 
diseases. In accordance with tradition, he de- 
livered the Heberden Oration in London. Dr. 
Bunim was also selected to give the Stoneburner 
Lecture in Richmond, Va., as guest lecturer for 
the Mexican Rheumatism Society in Mexico City 
and for the Canadian Rheumatism Association in 
Toronto. He delivered lectures on the rheumatic 
diseases at London University, Manchester Uni- 
versity and Leiden University. Dr. Paul A. di 
Sant'Agnese was appointed Clinical Professor of 
Pediatrics at Georgetown University and Lec- 
turer in Pediatrics at Johns Hopkins University. 
He was given the annual award of the Philadel- 
phia Chapter of the National Cystic Fibrosis 



Research Foundation. Dr. di SantAgnese was 
guest lecturer at scientific meetings in Holland, 
Switzerland and Germany. 

At the request of the Bureau of Medical 
Services of the U.S.P.H.S., members of the 
Clinical Investigations staff gave lectures and 
conducted ward rounds at the following PHS 
Hospitals : Baltimore, Boston, Seattle and Staten 
Island. 

Our staff scientists have derived substantial 
benefit from the association of distinguished 
Visiting Scientists and Guest Workers who have 
worked at our Institute during the past year: 
Prof. E. G. L. Bywaters of London University, 
Dr. Rosalind Pitt-Rivers of the National Insti- 
tute for Medical Research, England, Dr. Samuel 
Rose of the University of Melbourne, Australia, 
Dr. Serge Lissitzky of the University of Mar- 
seilles, France, Dr. P. Vilkki of the University 
of Turku, Finland, Dr. H. Keen of Guy's Hos- 
pital, England, Dr. Marco Andreoli of the Uni- 
versity of Rome, Dr. T. Shiba of Osaka Univer- 
sity, Japan and Dr. David Jackson of the Uni- 
versity of Oregon. 

Arthritis and Rheumatism Branch 

Research activity in the immunological reac- 
tions in the rheumatic diseases is increasing 
sharply both in this country and abroad. This 
shift in research direction is understandable since 
studies conducted on the chemistry and metabo- 
lism of connective tissue over the last decade have 
not achieved a clear insight into the pathogene- 
sis of the rheumatic diseases. Newer concepts 
and knowledge of the mechanism of antibody 
(both humoral and cell-bound) formation and 
more sophisticated immunological techniques 
have stimulated workers in the rheumatism field 
to renewed efforts in this relatively young and 
rapidly growing discipline. 

Serological Factors in Connective Tissue Diseases 
As reported last year, it was discovered in this 
laboratory that patients with Sjogren's syndrome 
(kerotoconjunctivitis sicca, xerostomia and rheu- 
matoid arthritis or other connective tissue dis- 
ease) exhibited an unusually high incidence of 
circulating "antibodies" or "factors" to tissue 
components which by and large were not organ 
or species specific. (These studies have been ex- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



177 



tended during 1960 and interesting results ob- 
tained). The series now includes 40 patients with 
Sjogren's syndrome that may be conveniently 
classified into four clinical groups: (1) sicca 
complex with rheumatoid arthritis, (2) sicca 
complex with scleroderma, (3) sicca complex 
with polymyositis or myopathy and (4) sicca 
complex alone. The greatest number of separate 
antibodies and the highest titers of these were 
present in the last two groups, yet the incidence 
of different antibodies even in the first two groups 
clearly exceeded that found in rheumatoid ar- 
thritis alone. The incidence of individual sero- 
logical factors demonstrated in Sjogren's syn- 
drome were: rheumatoid factor, 100 percent; an- 
tinuclear factor, 77 percent; complement fixing 
antibodies against tissue components, 49 percent 
and specific thyroglobulin antibodies, 27 percent. 

By the Ouchterlonly gel diffusion precipitin 
technique, precipitin lines were demonstrated be- 
tween serum gamma globulin fractions from pa- 
tients with Sjogren's syndrome and saline ex- 
tracts from human salivary gland, liver, kidney 
and thyroid extracts. Attempts to isolate the 
active (antigenic) fraction concerned in the com- 
plement fixation reaction were made by ultra- 
centrifugation of tissue homogenates. The anti- 
genic component was found in the soluble (super- 
natant) constituent and not in the sedimented 
nuclei, mitochondria or microsomes. The serum 
constituent involved in this reaction was found 
in the gamma globulin fraction following am- 
monium sulfate and electrophoretic separation. 

Of special clinical interest was the observation 
of the concurrence of Hashimoto's thyroiditis, 
arteritis, myopathy, hyposthenuria and neuro- 
pathy in some cases in this series. 

The results of these studies suggest that an 
altered or deranged immunological mechanism 
may be involved in the pathogenesis of Sjogren's 
syndrome which is frequently associated with 
rheumatoid arthritis or some other connective 
tissue disease (scleroderma, systemic lupus ery- 
thematosus or polyarteritis nodosa). 

Action of Steroids on Enzyme Systems 

Inhibition of DPNH-Cytochrome C Reductase. 
As described in the 1959 Annual Report, low 
concentration of hormonally active steroids and 
diethylstilbestrol were shown to inhibit DPNH- 



cytochrome c reductase between the flavoprotein 
and cytochrome b (or Coenzyme Q), and this 
inhibition could be competitively overcome by 
a-tocopherol and various other lipids. Further- 
more, an alternate route of electron transport 
was identified from the flavoprotein to cyto- 
chrome c. As a future step in localizing the 
effect of steroids on this sequence (as described 
in earlier reports) the reduction of internal co- 
enzyme Q was studied in beef heart mitochon- 
dria. As expected, steroid inhibition of Co Q 
reduction was observed, thus confirming the site 
of inhibition to be just beyond the flavoprotein 
level.* 



(DPNH y FP 




>C- 



-M 



Additional work has been done in characteriz- 
ing the basic effect, and particularly in identify- 
ing possible physiologic sequelae. It has been 
shown that soluble preparations of cytochrome c 
reductase from microsomes and mitochondria are 
affected in a manner similar to the particulate 
preparations. Some specificity of the lipid re- 
versal of the steroid effect is suggested since a-to- 
copherol does not diminish the degree of inhibi- 
tion of DPNH oxidation by amytal (or chlor- 
promazine) both of which block the electron 
transport chain at approximately the same level. 
More intensive study of beef pituitary and human 
placental enzyme preparations has not revealed 
any striking differences in response from the 
rat tissues examined. 

Decarboxylation of Pyruvic Acid. The DPN- 
dependent oxidation of pyruvic acid to acetyl co- 
enzyme A and C0 2 is the metabolic step which 
permits the entry of glucose carbon into the 
Krebs cycle, and its rate might be important in 
the regulation of carbohydrate metabolism. 
Steroid hormones have been shown to inhibit this 
reaction in vitro; and the elevated blood levels of 
pyruvic acid in patients with Cushing's disease 
and upon steroid administration to normals 9 
suggest some such action of these hormones in 
vivo. 

Since the oxidation of DNPH to DPN by the 
DPNH-oxidase reaction is strongly inhibited by 
various steroid hormones, it was suspected that 



178 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the steroid inhibition of pyruvate oxidation might 
be due to curtailment of DPN arising from 
DPNH oxidation. 

Pyruvate oxidation in homogenates of rat tis- 
sue was stimulated by the addition of cytochrome 
c and/or liver microsomes, which specifically 
stimulate DPNH oxidation. This showed that 
the rate of pyridine nucleotide oxidation could 
control that of pyruvate. Alpha-tocopherol, 
which prevented steroid inhibition of the DPNH 
oxidase, also overcame the steroid suppression 
of the pyruvate reaction. When DPNH oxida- 
tion was impeded by other inhibitors of electron 
transport, such as amytal or antimycin A, there 
was a corresponding depression of the oxidation 
of pyruvate, and a-tocopherol could not over- 
come this effect. 

These findings illustrate that steroids can regu- 
late the oxidative decarboxylation of pyruvic 
acid, by virtue of their ability to inhibit oxida- 
tion of DPNH. The possibility is being investi- 
gated that a direct mechanism of inhibition also 
exists. 

It has been established by other investigators 
that steroid hormone administration can result in 
a five-fold increase in GPT activity in the rat 
liver. In order to examine the possibility that 
this induction could result from the accumula- 
tion of substrate pyruvate after steroid admin- 
istration, a group of six rats was given injections 
of pyruvate over a 2-week period. Comparison 
of the liver GPT activities with six controls 
revealed no significant differences. The question 
of whether the pyruvate was actually increased 
at the cellular level by this procedure has not 
been established. 

Inhibition of Glutamic Dehydrogenase (GDH) 
Reaction. In studying the possible sequelae of 
the steroid inhibition of DPNH oxidation, we 
were led to examine the effects of steroids on cer- 
tain DPN requiring dehydrogenases. The oxi- 
dation of glutamate to a-ketoglutarate (<*KG) 
and ammonia by DPN (or TPN) the GDH re- 
action was of particular interest because of the 
importance of glutamic acid in amino acid syn- 
thesis and breakdown, it seemed reasonable in a 
system which contains both glutamic dehydro- 
genase and DPNH-cytochrome c reductase, the 
inhibition of the latter reaction by steroids could 
depress the oxidation of glutamate when DPN 



was rate-limiting. In accordance with this, 5 x 
10" 5 M progesterone caused a 2.7-fold increase 
in the rate of DPNH accumulation in a system 
consisting of DPN, gluamate and disrupted liver 
particles. 

When this phenomenon was examined in 
greater detail, however, it was found that be- 
sides their effect on DPNH-cytochrome c re- 
ductase, some steroids could inhibit glutamic 
dehydrogenase directly. This was shown with 
soluble extracts prepared by freezing and thaw- 
ing liver mitochondria in distilled water. Sub- 
sequently, the reaction has been studied in con- 
siderable detail, using the crystalline enzyme 
prepared from beef liver. When the activity is 
assessed either by reduction of a-ketoglutarate or 
oxidation of glutamic acid, using either di- or tri- 
phosphopyridine nucleotide as co-factor, various 
steroids (and diethylstilbestrol (DES)) were 
able to produce significant inhibition. 

In addition to detailed kinetic appraisal of the 
steroid-enzyme interaction, evidence has been ob- 
tained that the steroids induce a reversible change 
in the structure of the enzyme. 

Diethylstilbestrol, estradiol, progesterone and 
testosterone (in that order), were the most effec- 
tive compounds studied, while the corticoids were 
relatively ineffective either alone or in combina- 
tion with other steroids. Diethylstilbestrol pro- 
duced significant inhibition in concentrations as 
low as 10" 7 M. Of considerable interest was the 
fact that adenosinediphosphate (ADP) (and to 
a much lesser extent adenosinetriphosphate 
(ATP) and adenosinemonophosphate (AMP) ) 
prevented the steroid inhibition, particularly 
since 10 times as much ADP was necessary to re- 
verse the inhibition observed when the reductive 
reaction was studied. 

A study of steroid concentration effects, both 
in rat liver mitochondria and in the crystalline 
beef liver enzyme, has yielded complex data per- 
haps best explained on the basis of multiple 
binding sites for the steroid. 

The steroid inhibition has been extensively ex- 
amined using varying concentrations of the dif- 
ferent substrates (glutamate, DPN, TPN, DPNH, 
TPNH, a-ketoglutarate, NH 3 ) and no clear-cut 
competitive relationships have been established. 
However, very high (Ca 10" 2 M) concentrations 
of DPN, but not TPN, will prevent the steroid 
effect. Conversely, high concentrations of a-keto- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



179 



glutarate, DPNH, and TPNH, enhance the inhi- 
bition. The complicated kinetics of the reaction 
in the presence of steroid suggest that at least 
one form of the enzyme-inhibitor complex may 
retain some catalytic activity. 

Some information has been obtained concern- 
ing the binding site of the steroid, suggesting 
that a sulfhydryl group may be involved. Para- 
chloromercuribenzoate (PCMB), in concentra- 
tions insufficient to inhibit the enzyme, was shown 
to reduce the degree of steroid inhibition. Ap- 
propriately, DES was also shown to retard the 
formation of enzyme-PCMB complex, as meas- 
ured by following the optical density of the com- 
plex at 250 mju.. On the other hand, the steroids 
were shown to prevent I 2 inactivation of the 
enzyme. 

In addition to the beef and rat liver enzymes, 
GDH has also been prepared from chicken liver, 
rat testis, rat kidney, rat heart, S.typhrimurium 
and Neurospora crasa. Only the latter two failed 
to show steroid inhibition. 

Thyroxine and 1,10-phanthroline, both inhibi- 
tors of the GDH reactions, were additive with 
steroid. 

Since it had been shown by Frieden that vari- 
ous inhibitors of GDH could dissociate the en- 
zyme into inactive subunits, the effects of steroids 
and DES on the sedimentation properties of the 
enzyme were examined. It was observed that 
DES, estradiol, and progesterone could promote 
a dissociation of the crystalline enzyme into two 
forms appearing in the ultracentrifuge as two 
peaks of 12-14s and 24-26s. The effectiveness of 
the three compounds as inhibitors of the reaction 
was well correlated with the degree of splitting 
observed. ADP, which prevented the inhibition 
of the reaction, also prevented the steroid dis- 
sociation of the enzyme. 

Further evidence for competition between 
PCMB and steroid was obtained in that the 
PCMB-inactivated enzyme could not be split by 
steroid. 

The intimate mechanism by which certain 
steroids can dissociate such a protein aggregate 
into subunits has not been determined. Although 
GDH is rich in sulfur, it is apparently not held 
together by S-S bonds since prolonged incuba- 
tion of the enzyme with the reducing agent, thio- 
glycollate, did not result in any decrease in ac- 
tivity. (In fact, thioglycollate protected GDH 



against loss in activity from prolonged incuba- 
tion and prevented steroid inhibition.) It is per- 
haps revealing that GDH could also be dissoci- 
ated with the detergent, sodium dodecylsulfate, 
although it was somewhat less effective than the 
steroids. A possible mechanism, then, is that of 
hydrophobic bond disruption. 

The interesting findings of Struck and Sizer 
that GDH can catalyze the oxidation of amino 
acids other than glutamate (albeit at considerably 
slower rates) prompted an examination of the 
effects of steroids when these are used as sub- 
strates. It has been observed that steroids pro- 
duce varying effects, depending on the particular 
substrate used. Of greatest interest was the re- 
versible reductive amination of pyruvate to ala- 
nine which can be catalyzed by GDH at a rate 
1/200 that of glutamate. That the same protein 
is responsible for the two activities was proved 
by DEAE cellulose column chromatography, heat 
inactivation, alcohol fractionation, and ultracen- 
trifugation. Evidence indicates that the pyru- 
vate-alanine interconversion is catalyzed chiefly 
by the dissociated form of the GDH molecule 
which is inactive toward glutamate. Thus the 
pyruvate-alanine reaction is stimulated by steroid 
hormones, DNPH, and 10,phonarthroline, all of 
which dissociate GDH into subunits and thus in- 
hibit glutamate oxidation. On the other hand, 
the alanine pyruvate activity was inhibited by 
those things such as high concentration of DPN 
and TPN and ADP which cause association of 
enzyme and stimulate glutamate oxidation. 

Thus, not only does the state of aggregation of 
the GDH molecule determine its catalytic be- 
havior as Frieden has shown, but also its sub- 
strate specificity. Therefore, steroid hormones 
in dissociating glutamic dehydrogenase into sub- 
units, impede its function in interconverting glu- 
tamate and a-ketoglutarate, while facilitating its 
function in catalyzing the alanine dehydrogenase 
reaction. 

The present findings hopefully may lead to a 
better understanding of the mechanisms of hor- 
mone action. Although the physiological impact 
of such an effect on the glutamic dehydrogenase 
reaction cannot be appraised at the present time, 
these findings present an interesting model sys- 
tem of steroid action in which both the catalytic 
behavior and substrate specificity of an enzyme 
are changed as a result of a steroid-induced al- 



180 



ANNUM, REVIEW OP INTRAMURAL RESEARCH 



teration in the structure of the enzyme molecule. 
In addition, the finding that a single enzyme may 
catalyze different reactions, depending on its state 
of aggregation, presents a model mechanism by 
which both diversification of enzyme function and 
development of new genetic characteristics could 
occur. 

Purine Metabolism in Gout 

The cause of the hyperuricemia of gout, 
whether increased production or diminished ex- 
cretion of uric acid, has been studied further. 
The extent to which an excessive production of 
uric acid contributes to the hyperuricemia of 
gout has been evaluated by administering gly- 
cine-l-C 14 , a metabolic precursor of uric acid, 
along with uric acid-N 15 to normal and gouty 
subjects and determining the recovery of isotope 
in urinary uric acid. The data thereby obtained 
provide two independent parameters of uric acid 
synthesis: the extent to which glycine is incor- 
porated into uric acid and the size of the body 
urate pool and its turnover. In addition, the 
fraction of the daily uric acid production that 
is disposed of by routes other than renal excre- 
tion (uricolysis and deposition in tophi) is indi- 
cated by the percent of the administered uric 
acid-N 15 which is recovered in the urinary uric 
acid. Nine of 16 gouty patients showed an in- 
corporation of glycine-1-C 14 into urinary uric 
acid ranging from only slightly above normal 
to several times normal. In two of the seven 
remaining patients an abnormally large extra- 
renal disposal of uric acid masked an increased 
glycine incorporation. There remained five gouty 
subjects in whom no evidence of an increased 
uric acid production could be found by either 
glycine incorporation data or urate turnover 
values. 

The extent to which an impaired renal handling 
of uric acid contributed to the hyperuricemia of 
this group of gouty subjects was investigated by 
determining the urate/inulin clearance ratio. For 
comparison clearance values were obtained in 
nongouty subjects before and after uric acid pro- 
duction was increased by feeding ribose nucleic 
acid (RNA). The group of five gouty subjects 
with a normal uric acid production showed a 
lower urate/inulin clearance value than was ob- 
tained with normal subjects who were receiving 
RNA. Five of the six patients who produced 



excessively large amounts of uric acid showed 
urate/inulin clearance ratios in the same range 
shown by nongouty subjects whose uric acid pro- 
duction had been increased by feeding UNA. 

It would seem unlikely that a single inborn 
metabolic defect characteristic of all gouty pa- 
tients would produce in some individuals an 
excessive uric acid production and in others no 
increase in uric acid production but instead a 
diminished ability of the kidney to dispose of 
uric acid. We are led then to the view now be- 
ing explored that clinical gout may be the result 
of a variety of basic metabolic or physiological 
disturbances which have in common the induction 
of a hyperuricemia. 

Aromatic Amino Acids 

Objectives in studying patients with metabolic 
diseases associated with the metabolism of the 
aromatic amino acids, alcaptonuria, phenylke- 
tonuria, tyrosinosis and albinism, have been sev- 
eral: (1) to determine the exact nature of the 
metabolic defect in these conditions; (2) to study 
the hereditary pattern of these diseases and, if 
possible, to develop tests which will detect the 
heterozygous state in relatives carrying the trait; 
(3) to study the formation and deposition of the 
pigment derived from homogentisic acid and to 
determine how it produces the pathological 
changes in the connective tissues, particularly the 
joints (ochronotic arthritis) ; (4) to study the 
cause of ochronotic arthritis, nearly always as- 
sociated with alcaptonuria; and (5) to attempt 
various means of treatment of these metabolic 
diseases. 

Ascorbic Aero in Tyrosine Metabolism. Our 
studies have continued on the detailed mechanism 
by which vitamin C maintains normal tyrosine 
metabolism through protecting p-hydroxyphenyl- 
pyruvic acid oxidase from inhibition by its sub- 
strate. Several analogues of ascorbic acid, such 
as D-isoascorbic acid, have also been found to 
protect this enzyme from inhibition in scorbutic 
guinea pigs in vivo. In addition, a dye, 2,6-di- 
chlorophenolindophenol, and folic acid also pro- 
tect the enzyme in scorbutic guinea pigs, and 
the action of the latter two compounds has been 
studied in more detail. The dye has been found 
to have no anti-scorbutic effects in vitamin C- 
deficient animals except for its ability to maintain 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



181 



tyrosine metabolism. Therefore, there is a clear 
distinction between the effect of vitamin C in 
tyrosine metabolism and the effects of the vita- 
min in the other aspects of scurvy. Folic acid 
was found not to protect p-hydroxyphenylpy- 
ruvic acid oxidase in vitro and we have concluded 
that its beneficial effect in vivo are through some 
indirect mechanism. 

Structural analogues of p-hydroxyphenylpyru- 
vate are being tested to find out the structural 
requirements for inhibition. In addition, a 
kinetic analysis of substrate-induced inhibition 
and that by other analogues has been made. 
Phenyl pyruvate has been found to be even more 
potent an inhibitor than the substrate when pre- 
incubated with p-sydroxyphenylpyruvic acid oxi- 
dase and the inhibition is of the non-competitive 
type under these circumstances. Various other 
dyes and redox systems are being tested in place 
of 2,6-dichlorophenolindophenol to determine 
whether it is a structural or redox property of 
the dye which is the important component in its 
effectiveness in protecting the enzyme. Recent 
results indicate that it is the structure and/or 
the ease of its reduction, rather than the E 
value of the redox system which is important. 
The compounds active like the dye also have in 
common the ability to undergo a one electron 
change in oxidation, suggesting that a free radical 
may participate in the oxidation of the substrate 
by p-hydroxyphenylpyruvic acid oxidase. 

New Tome Effect of Prolonged Corticosteroid 
Therapy — Posterior Subcapsular Cataract 

During the past year we have observed and 
reported a heretofore unknown adverse effect of 
prolonged corticosteroid therapy in high dosage, 
namely posterior subcapsular cataracts. Although 
many of the patients observed to have such cata- 
racts had been receiving dexamethasone, a large 
number of others had never received this specific 
analogue, but had received both the naturally 
occurring and the older synthetic corticosteroid 
preparations. Seventeen of 44 rheumatoid ar- 
thritis patients having received corticosteroid 
therapy for periods of one year or longer were 
found to have this ocular lesion. Nineteen non- 
steroid treated rheumatoid patients were exam- 
ined and no such lesions were found. The ap- 
pearance of the posterior subcapsular cataract 
was found to be associated with moderate or 



high dose therapy (equivalent to 10 mgm. or 
more of prednisone daily) for a period of one 
year or longer. More recently, an additional 35 
corticosteroid-treated patients have been exam- 
ined, and 8 of these individuals were found to 
possess this lenticular opacity. Among these 8 
were two young boys, ages 8 and 12. The grad- 
ual improvement with decrease in size of opacity 
in two patients in whom it was possible to re- 
duce or completely discontinue therapy has sug- 
gested, in some individuals at least, that this 
lesion may be reversible. Lenses have been ob- 
tained from one patient at autopsy and from 
another at surgery, although the microscopic 
findings are not available at the time of this re- 
port. The correlation between steroid adminis- 
tration and the appearance of posterior subcap- 
sular cataracts is highly significant (0.01 > P 
> 0.001). Other potential factors of etiologic 
significance of cataract formation were carefully 
assessed, including gold therapy, salicylate the- 
rapy, X-ray exposure, and estrogen therapy, as 
well as calcium intake. No significant correla- 
tion was shown between these factors and the 
appearance of cataracts. Scientists in the Oph- 
thalmology Branch of the NINDB cooperated in 
these studies. 

New Antirheumatic Drugs 

6-Alpha Fluorotriamclnolone. A second meta- 
bolic study involving this compound administered 
in dosages of 20 mgm. has now been completed 
by Dr. G. Donald Whedon and his group. One 
year ago metabolic studies of this compound were 
carried out with a patient with rheumatoid ar- 
thritis. The data revealed the maintenance of a 
positive calcium balance during the administra- 
tion of 20 mgm. of this steroid daily. The second 
metabolic study completed during the year 1960 
was performed upon a normal control patient, 
again at a dosage of 20 mgm. daily. In this in- 
stance a slightly negative calcium balance was 
induced. This compound, although only 1/2 to V3 
as potent as dexamethasone in anti-inflammatory 
activity deserves further investigation in regard 
to its effect on calcium metabolism. 

Hydroxychloroquine. In cooperation with the 
American Rheumatism Association's Working 
Committee on Cooperative Clinics, the NIH- 



182 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Georgetown University Rheumatology Service is 
participating as a member of the committee in 
therapeutic trials of hydroxychloroquine in rheu- 
matoid arthritis. The entire enterprise embraces 
the acivity of 10 arthritis clinics located in vari- 
ous University centers throughout the country. 
The first clinical trial of hydroxychloroquine be- 
gan in February of 1960 and was completed in 
July of the same year. Each patient in the trial 
was observed over a 3-month period. The trial 
was conducted as a double-blind study on a total 
of 76 patients. The parameters of arthritis ac- 
tivity included the evaluation of joint tenderness, 
grip strength, ability to walk and the erythrocyte 
sedimentation rate. A statistically significant 
difference was demonstrated between patients re- 
ceiving the compound and those receiving pla- 
cebo. Differences between the two groups of pa- 
tients were observable as early as one month after 
the institution of therapy. No significant side 
effects were observed during the 3-month period. 
It is anticipated that another trial will be insti- 
tuted within the next 3 months. 

An Anti-Metabolic Agent. A new evaluation 
of an antimetabolic agent has been undertaken in 
patients with psoriatic arthropathy. A double- 
blind study has been carefully planned. To date 
four, of a planned series of 40 patients, have 
been included in the trial. Each patient will be 
observed over a period of 60-80 days on therapy. 
During this period the patients will be on a 
standard diet. Careful observations of joint in- 
flammation as well as skin involvement will be 
made. The study is being conducted in coop- 
eration with Dr. Eugene Van Scott and Dr. 
Arthur Eisen of the National Cancer Institute. 



Gastroenterology 

In keeping with the objective of studying the 
biochemistry and morphology of the small intes- 
tine of man, the gastroenterology group has set 
up a large number of chemical tests and other 
procedures for the study of intestinal absorption. 
These have been applied in the clinical projects 
described below. A laboratory study of bile pig- 
ment metabolism has also been started and an 
enzyme not previously isolated, has been partially 
purified. 



Effects of Radiation and Folic Acid Antimetabo- 
lites on Intestinal Absorption 

The small bowel mucosa is extremely sensitive 
to radiation injury. Animal studies have shown 
that exposure to radiation can produce not only 
marked morphological changes, but also altera- 
tions in absorptive functions. To explore these 
observations in man, a collaborative study has 
been initiated with investigators in NCI. Pa- 
tients who are to receive radiation as cancer 
chemotherapy are evaluated before and after 
radiation exposure by assessments of intestinal 
absorptive capacities and by small bowel mucosal 
biopsy. Three patients have been studied so far 
and a much larger series is contemplated. The 
information obtained in this study will be impor- 
tant not only in relation to human intestinal 
physiology, but also in relation to the effects of 
radiation on man. 

Similarly, animal studies have shown that in- 
terference with folic acid metabolism can pro- 
duce morphological and functional changes in the 
small intestine. Again in collaboration with 
NCI, a study is in progress to evaluate intestinal 
\function before and after patients with psoriasis 
receive methotrexate therapy. 

Malabsorption and Osteoporosis 

A long-standing project in NIAMD has been 
devoted to studies of osteoporosis. In collabora- 
tion with the investigators working on this prob- 
lem, a preliminary survey has been made to de- 
termine whether osteoporotic patients might be 
suffering from clinically unsuspected steatorrhea, 
a condition that might cause a drain on their 
dietary calcium. Of five patients studied, four 
proved to have significant steatorrhea. Studies 
by the gastroenterology group suggested that the 
steatorrhea was not attributable to disease of the 
small intestine. Future studies will be directed, 
therefore, at the possibilities that pancreatic or 
biliary disease might explain the steatorrhea. 
The problem of how the steatorrhea may be im- 
plicated in the osteoporosis will also be investi- 
gated. 

Juvenile and Adult Celiac Disease 

Evidence of recent years from other labora- 
tories suggests that celiac disease of children and 
nontropical sprue of adults are attributable to a 
single underlying disorder, that the disorder is 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



183 



hereditary, and that it predisposes an affected 
individual to an injurious action of such proteins 
as wheat gluten. This theory is under evalua- 
tion by the gastroenterology group. Patients 
are admitted for extensive screening to determine 
whether they are susceptible to the toxic action 
of wheat protein. When a positive diagnosis is 
made, as many of the patient's blood relations as 
are available are admitted to the Clinical Center 
in order to study the genetics of celiac disease. 
Previous studies of the genetics of celiac disease 
have relied only on medical histories of relatives 
of affected individuals. In the present study 
careful chemical evaluations of intestinal absorp- 
tion are carried out and intestinal biopsies are 
taken. Six members of a single family have 
been studied in this way. Additional families 
will be added to the series as they are referred. 
Additional use is made of the biopsy material 
obtained in this project. Together with Dr. 
Samuel Spicer, NIAMD, the specimens are sub- 
jected to histochemical studies of their mucopro- 
teins. Information about the mucoproteins of the 
intestinal mucosa of normal man is extremely 
scanty and even less is known about disease 
states. Tissues from 20 patients have been ob- 
tained during the past year and are now under 
etudy. 

Protein Metabolism in Malabsorption States 

Malabsorption of various etiologies is often 
associated with low levels of serum proteins. It 
has been assumed that the deficiency of these 
proteins is due to impairment of absorption of 
the protein building blocks and consequent sup- 
pression of protein synthesis. Together with 
Dr. Thomas Waldmann, NCI, patients with mal- 
absorption on the gastroenterology service are 
being studied to characterize their protein me- 
tabolism. Four patients of a clinically unusual 
type, have been studied so far. In some there 
was evidence of impaired protein biosynthesis, 
but in others the protein deficiency appeared at- 
tributable to loss of protein by leakage from the 
gastrointestinal tract. A large series of such 
cases will be studied as appropriate patients be- 
come available for study. 

Mammalian Metabolism of Bile Pigments 

Bile pigments, derived from the heme of hemo- 
globin, are processed in the liver, excreted via 



the bile into the small intestine and eliminated 
in the feces. A portion of the intestinal bile 
pigments is reabsorbed, returned to the liver and 
reexcreted. The enzymatically catalyzed reac- 
tions that occur during this enterohepatic circu- 
lation of bile pigments have not been studied in 
detail. Such a study has been initiated in this 
laboratory and the first enzyme in this important 
pathway of metabolism has been partially puri- 
fied and many of its properties have been deter- 
mined. After studies of this enzyme are com- 
plete, the next one in the sequence will be ex- 
plored. 

Clinical Endocrinology Branch 

Carbohydrate Metabolism 

Glucose. It has been known for some time that 
mammalian liver contains enzymes capable of 
effecting oxidation of the first carbon atom of 
glucose via a pathway known as the hexose 
monophosphate shunt (HMP). The quantita- 
tive significance of this pathway in the intact 
human has not, however, been determined. Meas- 
urement of C 14 2 and blood glucose C 14 after 
the administration of Ci 14 glucose and C 8 14 glu- 
cose have been made in normal human subjects. 
It was found that data for the kinetics of carbon 
labeled bicarbonate excretion were also necessary 
for this formulation. In collaboration with Dr. 
M. Berman of the Biomathematics panel, a sat- 
isfactory analysis of the rather complex excretory 
patterns was obtained. The results show that in 
normal man about 10 percent of the total glucose 
is oxidized via the shunt pathway. Preliminary 
data indicate that this fraction is decreased in 
hyperthyroidism. The functional importance of 
the shunt pathway which cannot be evaluated 
from enzyme content of isolated tissues can now 
be determined in various diseases. 

Glucose metabolism has also been studied in 
isolated tissues. In the isolated rat diaphragm 
it was shown that the transport of 3-deoxyglu- 
cose into the cell is unaffected by insulin, and 
does not furthermore inhibit glucose transport 
nor interfere with glucose oxidation. A rather 
extensive series of studies have been conducted 
on the metabolism of glucose in isolated endocrine 
tissues; in particular, evaluation of the extent 
of the shunt pathway utilizing carbon one and 
carbon six labeled glucose has been performed. 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



It has been shown that in all endocrine tissues 
studied a very active hexose monophosphate 
pathway is present. The tissues studied include 
the adrenal, ovary, testis, anterior pituitary, 
parathyroid and thyroid. The thyroid has been 
particularly carefully studied. It has been pre- 
viously reported from this section that thyroid 
stimulating hormone (TSH) from the pituitary 
in vitro caused marked stimulation of the oxida- 
tion of glucose-1-C 14 and to a lesser extent the 
oxidation of glucose labeled 6-C 14 . This stimu- 
lation is manifest 5 minutes after the addition 
of TSH and is effective at a concentration of 
3 x l(h 11 M. The mechanism of this effect has 
been investigated and it appears likely that it 
is due to an increased synthesis of triphospho- 
pyridine nucleotide from diphosphopyridine nu- 
cleotide, since TSH causes a rise in TPN and a 
concomitant fall in DPN, although the levels of 
TPNH and DPNH are not significantly changed. 
It has not yet been possible to demonstrate these 
effects in cell free systems. Other agents have 
also been found which effect glucose metabolism 
in thyroid slices. Quite low concentrations of 
acetylcholine stimulate both C-l and C-6 labeled 
glucose oxidation and increase glucose uptake. 
Atropine inhibits the acetylcholine effect with- 
out altering the TSH effect. There is also par- 
tial inhibition of the acetylcholine stimulation 
by large amounts of iodide. Epinephrine and 
serotonin have also been shown to stimulate glu- 
cose oxidation by thyroid slices. This effect can 
be observed in cell free systems fortified with 
TPNH. TSH has also been shown to stimulate 
the oxidation of labeled pyruvate and acetate by 
thyroid slices in vitro. 

Pancreatic islet cell tissue has been studied in 
islet cell tumors removed from individuals and 
in the islet cell tissue of certain fish in which 
this tissue is present as a discrete tissue uncon- 
taminated by pancreatic exocrine glandular tis- 
sue. In all these specimens of islet tissue an 
active hexose monophosphate pathway was dem- 
onstrated. In the tumors it was shown that 
alloxan did not influence glucose oxidation al- 
though in one case leucine and orinase appeared 
to stimulate oxidation. The oxidation of glucose 
by islet tissue is extremely rapid, exceeding, on 
a weight basis, that observed in either liver or 
heart. 

The oxidation of glucose by ovary and testis 



was unaffected by the trophic hormones, FSH, 
and LH, although in both ovary and testis an 
active shunt pathway was obtained. The pres- 
ence of a shunt pathway was also demonstrated 
in the anterior pituitary and it was shown that 
serotonin and catechol amines stimulated the 
formation of carbon dioxide from one labeled 
glucose. The glucose metabolism in adrenal tis- 
sue was studied and an active shunt pathway 
was demonstrated. Interestingly enough neither 
ACTH nor cyclic 3'5' AMP had an effect on 
oxidation of glucose. 

Metabolism of glucose by the mammary gland 
has also been studied. Prolactin, growth hor- 
mone and ACTH have all been shown to be 
active in stimulating the oxidation of carbon 
labeled glucose by slices of rat lactating mam- 
mary glands. The same hormones have also 
been found to increase the glucose uptake in 
mammary gland. Glands obtained at the end 
of lactation were found to be extremely respon- 
sive to these hormones and as little as 5 /*g were 
effective. 

Insulin. Studies on the metabolism of insulin 
have been continued and it has been demonstrated 
that in the isolated perfused rat liver insulin 
prevents the release of potassium into the per- 
fusate. During control perfusions in the course 
of 60 minutes, approximately 7i/ 2 percent of the 
liver potassium was released into the perfusate, 
with a proportional loss of water. The addition 
of insulin to the perfusing medium prevented 
both the potassium and the water loss. There 
appeared to be no significant effect of insulin 
on the net hepatic release of glucose. As was 
noted in last year's report, the cooling of the 
perfused liver inhibits the metabolism of insulin 
but does not interfere with binding of insulin by 
liver cells. To study further this phenomenon 
a fluorescent derivative of insulin has been pre- 
pared by the reaction of fluorescein isothiocyanate 
with crystalline insulin. A preparation so ob- 
tained has been shown to retain 60 percent of its 
hypoglycemic activity. Preliminary experiments 
have shown that this derivative is strongly bound 
to leucocytes and not to red cells. It will shortly 
be tested in the liver perfusion system. "Work 
has continued on assay of biological materials 
for insulinlike activity and several malignant 
tumors have been assayed. These tumors have 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



185 



been associated with hypoglycemia and in two 
of three studied, an extract was obtained which 
increased glucose uptake in the isolated dia- 
phragm. In one extract stimulation of glycogen 
deposition which was partially blocked with 
plasma containing insulin antibodies was noted. 
This rather bizarre apparent synthesis of insulin 
by nonpancreatic carcinomatous tissue is under 
further study. 

Galactose. Further studies have continued on 
the metabolism of carbon labeled galactose in 
individuals having the disease galactosemia. In 
collaboration with Dr. Topper, it has been found 
that menthol stimulates galactose oxidation in 
these subjects but not in normals. Hemolysates 
from galactosemic individuals failed to oxidize 
galactose and cannot be stimulated but hemoly- 
sates from individuals bearing the galactosemia 
trait show marked increase in galactose oxidation 
when pyruvate is added to the medium. This 
suggests that in the presence of even a small 
amount of transferase, stimulation of the 4-epi- 
merase (as with added pyruvate) accelerates the 
overall oxidation of galactose. Interestingly 
enough the accumulation of galactose-1-phosphate 
in leucocytes does not appear to alter the path- 
way of glucose metabolism in these cells. Galac- 
tose metabolism has also been studied in the rat 
and it has been shown that in the experimentally 
hyperthyroid rats there is an accelerated disposal 
of galactose. Rats fed a 30 percent galactose 
diet have been shown to have an amino aciduria 
quite similar to that seen in individuals with 
congenital galactosemia. The main amino acids 
excreted are taurine, alanine, aspartic and glu- 
tamic acid. Further studies have continued on 
the oxidation of galactose by human leucocytes 
and it has been shown that leucocytes from galac- 
tosemic individuals are virtually unable to oxi- 
dize galactose whereas leucocytes from individ- 
uals apparently harboring the galactosemia gene 
metabolize only approximately 50 percent of the 
galactose that normal individuals do. Thus it 
seems that individuals bearing this recessive trait 
can be identified by study of galactose metabo- 
lism in isolated leucocytes. In collaboration with 
Dr. Krooth of the National Cancer Institute, skin 
and bone marrow cells have been grown in tissue 
culture for up to 6 months. Galactosemic cells 
showed almost no growth when the sole carbo- 



hydrate source was galactose, whereas normal 
cells grew as well with galactose as with glucose. 
Galactosemic cells grown in glucose grew reason- 
ably well but after some months of growth were 
still unable to oxidize galactose to carbon dioxide. 
Further studies of carbohydrate metabolism on 
leucocytes from normal individuals, patients with 
glycogen storage disease and Hurler's syndrome 
have been performed. It has been shown that 
normal white cells have appreciable amounts of 
phosphorylase and glycogen but leucocytes from 
individuals with glycogen storage disease had 
unusually low levels of phosphorylase although 
the glycogen content was normal. Children with 
Hurler's syndrome, on the other hand, had un- 
usually low glycogen levels in their leucocytes. 
Further studies on these individuals are now 
under way to determine the defect in this syn- 
drome. 

Biochemistry of the Thyroid 

Iodide Transport. The iodide concentrating 
mechanisms of thyroid, mouse submaxillary, rat 
mammary, and rat thyroid tumor tissue slices 
has been studied. The tissue/medium ratio of 
I 131 (T/M) is greater for thyroid tissue than for 
all other tissues and in all four tissues is reduced 
to half the control value at from 1-3 x 10 -5 M 
iodide concentration. The difference in the T/M 
(I~) appeared to be a function of the capacity 
of the system rather than the affinity for iodide 
ion. In all four tissues iodide concentration was 
a function of the external potassium concentra- 
tion with half maximal stimulation attained at 
approximately 10"3M added potassium. The gly- 
cosides, ouabain and scilliroside depress the T/M 
ratio in all four tissues which depression can be 
partially reversed by the addition of potassium, 
rubidium, or cesium ions. Preliminary results 
show that there is a minimum size requirement 
in salivary and thyroid tissues for monovalent 
anion accumulation and ions of somewhat greater 
partial molar ionic volume than iodide such as 
pertechnetate and perrhenate show greater affin- 
ity constants for thyroid tissue than iodide. 
There is also a potassium requirement for the 
concentration of these ions and the inhibitor 
effects suggests that their accumulation occurs 
by a process similar to iodide concentration. 



186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Phospholipid Meetabolism. Phospholipid me- 
tabolism using P 32 has been studied in thyroid 
slices and homogenates. It has been shown that 
in thyroid slices P 32 uptake is stimulated by TSH 
and inhibited by digitalis glycosides. The inhi- 
bition by digitalis glycosides is largely overcome 
by increasing the potassium concentration in the 
medium. These effects are very similar to those 
seen with iodide concentration by the thyroid 
suggesting that there may be some relationship 
between phospholipid synthesis and iodide trans- 
port. The main phospholipid synthesized in re- 
sponse to TSH appears to be phosphatidyl in- 
ositol. Preliminary experiments have been per- 
formed attempting to extract a phospholipid 
from the thyroid which in in vitro systems will 
concentrate iodide. After extraction and chroma- 
tography on silica gel a lecithin like phospho- 
lipid has been obtained which will cause a small 
amount of iodide concentration under the follow- 
ing circumstances : Iodide is normally distributed 
between water and chloroform to the extent of 
several thousand times in favor of water. If 
this phospholipid extracted from the thyroid is 
present in the chloroform phase approximately 
equal partition between the two solvents is ob- 
tained. Chromatography of the radioactive io- 
dine in the chloroform phase shows it to behave 
as iodide. Cell free systems of thyroid gland 
incorporate almost no phosphate into phospho- 
lipids in spite of supplementation with a variety 
of cofactors. However an homogenate system 
can be obtained which will synthesize phospha- 
tide acid from P 32 labeled alpha-glycerol phos- 
phate. This step, however, does not appear to 
be effected by TSH, by potassium, or by digitalis 
glycosides. 

Iodoproteins. The iodoproteins of the thyroid, 
particularly thyroglobulin, have been the object 
of a rather extensive study in the past year. 
Thyroglobulin has been chromatographed on di- 
ethylaminoethylcellulose. It has been shown that 
thyroglobulin apparently homogenous with re- 
spect to sedimentation in the ultracentrifuge can 
be separated into three separate components on 
this ion exchange column. Preliminary studies 
have failed to disclose the chemical basis for the 
heterogeneity which is now under study. The 
iodination of thyroglobulin in vitro has been 
studied and it has been shown that in water at 
pH 9 only relatively small amounts of iodine are 



incorporated into tyrosine. In 8 Molar urea, 
however, or in detergent almost all the tyrosyl 
.groups appear to be equally reactive toward 
iodine, suggesting that a substantial proportion 
of the tyrosine residues are either buried in the 
protein or bonded in some way as to inhibit 
iodination at pH 9. This correlates reasonably 
well with the titration of thyroglobulin which 
shows that a substantial proportion of the tyro- 
sine groups in thyroglobulin have an abnormal 
pK being almost li/ 2 units more alkaline than 
the pK of free tyrosine. Recently a third type 
of tyrosyl group constituting about 15 percent 
of the total has been discovered. These tyrosyl 
groups do not begin to ionize until the pH of 
about 12% is reached and they then ionize at a 
rate which may be followed spectrophotometri- 
cally. Concomitant with these changes there are 
seen changes in the molecular size and a fairly 
symmetrical boundary in the ultracentrifuge oc- 
curs which has a sedimentation constant of about 
6. This is the minimum size so far observed in 
aqueous media for this protein. In collaboration 
with Dr. R. F. Steiner of the Naval Medical 
Research Institute, thyroglobulin has been stud- 
ied with the polarization of fluorescence tech- 
nique. In this case, conjugation with dimethyl- 
aminoaphthalene sulfonyl chloride has been used. 

The effects of treatment with urea, guanidine 
or detergent are more profound than those result- 
ing from thermal or alkaline denaturation. In 
each case, the transition is gradual. In no in- 
stance was any evidence of a cooperative process 
obtained. In detergents, urea and guanidine the 
transition is essentially reversible. The loosen- 
ing of the macromolectular structure by the lat- 
ter reagents renders the disulfide bridges suscep- 
tible to the action of reducing agents. However, 
even after reduction considerable internal rigid- 
ity remains. 

More recently studies have been undertaken on 
the structure of thyroglobulin as revealed by its 
antigenic properties. It was found that a fully 
hydrolyzed preparation of thyroglobulin retained 
the ability to precipitate rabbit antibodies to 
whole thyroglobulin as determined in gel diffu- 
sion. This was true when trypsin, chymotrypsin 
or pepsin was used for hydroylsis. The hydro- 
lysate contained a mixture of components with 
an average sedimentation constant of 3 to 4. On 
prolonged hydroylsis even smaller fragments are 
obtained which are dialyzable and which have 



NATIONAL INSTITUTE OF ARTHRITIS. AND. METABOLIC DISEASES 



187 



the capacity, albeit minimal to precipitate rabbit 
antibodies and are capable of inhibiting the pre- 
cipitation of native thyroglobulin with antiserum. 
These active components have been fractionated 
using sephadex and dialysis and it now appears 
that some of them have a molecular weight be- 
low 10,000. 

Thyroxine and Iodotyrosine Synthesis. In 
order to study the structural features of the 
thyroxine molecular necessary for its biological 
activity a variety of thyroxine analogues have 
been synthesized. These include the following: 



HO 




CH 2 €HC 



CH 2 CHC 



,COOC=H B 



^COOH 



COOC=H 5 



"COOH 



An interesting synthesis of thyroxine which re- 
sults in a rather high yield has been studied. In 
this synthesis diiodotyrosine and its keto ana- 
logue, 3,5-diiodo-4-hydroxyphenylpyruvic acid 
(HPPA) are used. A number of experiments 
were carried out to determine the structural fea- 
tures of HPPA required in this synthesis. The 
requirement for a three carbon atom side chain 
and for a free keto group seem to be so far abso- 
lute. Furthermore, the acylamino acid in which 
HPPA is peptide linked to glycine is also not 
effective. Incubation in the presence of ninhy- 
drin in order to form the 3,5-diiodo-4-hydroxy- 
phenylacetaldehyde in situ inhibited the synthe- 
sis of thyroxine. Studies with both radioactive 
diiodotyrosine and radioactive HPPA have 
shown, at least in preliminary form, that one 
molecule of HPPA reacts with one molecule of 
DIT. Because of the yield and for other rea- 
sons, it had been suspected that perhaps HPPA 
might act only as a catalyst. It now seems to 
actually be incorporated into the thyroxine. In 
the process of this study, it was necessary fo 
investigate the synthetic methods for the prepa- 
ration of keto acid peptides. A variety of meth- 
ods used for peptide syntheses were investigated 
and all except the reaction of HPPA with benzyl 
carbamate seemed to require blocking of the 
enolic hydroxyl by some sort of protective group. 



Because of uncertainties concerning the quani- 
tation of relative amounts of iodinated tyrosines 
and thyronines in the thyroid and blood, reinves- 
tigation of this problem was undertaken using 
a method of isotopic equilibrium. This method 
was validated with the finding that calculated 
and analyzed iodine values for thyroid iodine 
and for blood iodine agreed relatively closely. 
In rats on a relatively generous iodine diet, the 
molar ratios of MIT, DIT, T 4 and T 3 in the 
thyroid were, respectively, 9, 15, 5, and 1. In 
the serum, it was shown that thyroxine ac- 
counted for approximately 95 percent of the 
serum iodine and triiodothyronine for approxi- 
mately 4 percent. Kinetic studies of metabolism 
of these amino acids and their volume of distri- 
bution coupled with the higher potency of tri- 
iodothyronine suggests that in the rat almost 
half of the metabolic activity of the thyroidal 
secretions is accounted for by triiodothyronine. 
For the analysis of iodothyronines high voltage 
electrophoresis has been investigated and several 
organic solvents, formamide-glycine, pyridine- 
aqueous ammonia, and dimethylformamide-aque- 
ous ammonia have been found to be very sat- 
isfactory for separation of iodotyrosines and 
iodothyronines. Also a method was developed 
for the detection of iodinated materials on paper 
chromatograms or electrophoretic runs in which 
the cerate arsenite reaction was used and alpha- 
phenanthroline ferrous sulfate was employed as 
an indicator. This is highly sensitive and rea- 
sonably specific. 

Hormone Transport in Blood. Studies of thy- 
roxine binding proteins have continued. An ex- 
planation for the ability of a variety of drugs to 
lower the serum PBI has been obtained. It has. 
been shown that salicylate and dinitrophenol will 
competitively inhibit thyroxine binding to the 
prealbumin moiety of human plasma. Two ad- 
ditional drugs, tetrachlorothyronine and diphe- 
nylhydantoin have been shown to compete with 
thyroxine for binding sites on the thyroxine 
binding alpho globulin. All four of these drugs 
cause a lowering of the serum PBI apparently 
by interfering with thyroxine binding so that 
the concentration of free thyroxine is elevated, 
hence its degradation accelerated and a new 
steady state in which the normal level of free 
thyroxine is reached whereby the, total thyroxine 
is lowered. Studies have been continued in col- 



188 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



laboration with Dr. Blumberg on two-dimen- 
sional paper and starch gel electrophoresis of 
thyroxine binding proteins and serum. The 
identity of band 1 as seen in starch gel with 
pre-albumin as demonstrated in electrophoresis 
in ammonium carbonate buffer has been estab- 
lished. The identity of the thyroxine binding 
globulin with band 4 in starch gel also seems 
fairly clear. Work has progressed on the isola- 
tion of a corticosteroid binding protein from 
serum. Chromatography on DEAE and Ca 
phosphate gel have reslted in over 100 fold 
purification. This binding protein runs just be- 
hind albumin on starch gel electrophoresis. 

Effect of Thyroxine on Isolated Enzyme 
Systems. Earlier work on inhibition of dehy- 
drogenases by thyroxine has been extended. The 
native molecule of glutamic dehydrogenase 
(GDH) has a sedimentation rate of — 26S. Thy- 
roxine causes dissociation into units of ~14S 
and inhibition of enzymatic activity. Other halo- 
genated phenols cause dissociation and inhibition 
in about the same ratio. ADP and 5'AMP re- 
verse the thyroxine induced inhibition and cause 
reassociation of GDH. The kinetics of inhibi- 
tion of thyroxine and TPN, DPN and ADP 
favor binding of thyroxine at the second site on 
the enzyme. Of considerable interest is the re- 
tention of action by the thyroxine treated (and 
hence dissociated) enzyme with norvaline as a 
substrate. Furthermore, the dissociated enzymes 
action on norvaline is inhibited by ADP. 

Metabolic Diseases Branch 

Mineral Metabolism 

Dietary Calcium in Osteoporosis. Metabolic 
studies from this Branch are continuing to pro- 
duce pertinent evidence of the important rela- 
tionship of dietary calcium intake to the patho- 
genesis and possible therapy of post-menopausal 
and senile osteoporosis, a disorder estimated to 
affect approximately one-fourth of all women 
over the age of 50 years. As the result of this 
work, the concept of altered bone metabolism in 
osteoporosis is being expanded toward recogni- 
tion of a dual set of influences, hormonal and 
nutritional, and the idea of a complex etiology 
is gradually being more widely appreciated. 
Prior metabolic balance studies by this Branch 



have demonstrated the highly significant positive 
relationship between the level of calcium intake 
in the diet and calcium storage in patients with 
this disease. Radioisotopic studies with calcium- 
45 in revealing a normal rate of deposition of 
calcium in new bone formation in osteoporosis, 
have pointed to bone resorption as an important 
mechanism by which demineralization develops 
in this disease, a mechanism previously accorded 
little significance. In illuminating the impor- 
tance of resorption, the studies have indicated 
the inadequacy of the long-held previous concept 
of the pathogenesis of osteoporosis solely as im- 
paired osteoid matrix formation resulting from 
gonadal-corticoid hormonal imbalance. 

Recent studies by this Branch continue pro- 
gressively to indicate the complex nature of the 
pathogenesis of osteoporosis, suggesting particu- 
larly at present different processes by which in- 
testinal absorption and utilization of calcium 
may be impaired. A small group of patients 
with osteoporosis has been found which do not 
store calcium well even when the intake of this 
mineral is raised to very high levels. The initial 
common finding is steatorrhea at a subclinical 
level. Collaborative studies are in progress with 
the Clinical Gastroenterology group of this In- 
stitute in an effort to characterize the steatorrhea 
in these patients, whether of intestinal mucosal 
origin or pancreatic, and investigation is being 
made of the interrelationships of fat and mineral 
intestinal absorption. Preliminary observations 
suggest in some of these patients an abnormality 
of sweat electrolyte excretion similar to that seen 
in the heterozygous carrier state of cystic fibrosis. 

Hormonal and Nutritional Influences on 
Bone Metabolism. Balance and radioisotopic 
studies of calcium accumulation and turnover 
are continuing in patients with a variety of bone 
diseases under the influence of a number of nu- 
tritional and hormonal factors. Recent refine- 
ment of isotopic techniques is now making it 
possible to evaluate isotope excretion curves for 
30 to 40 days after administration of a tracer 
dose, so that it is anticipated that these extended 
data will provide significant information on rates 
of bone resorption as well as on currently cal- 
culatable rates of "bone formation." Utilization 
or tolerance tests of the handling of intravenously 
administered citrate and calcium are also being 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



189 



employed for analysis of mineral dynamics. Cal- 
cium tolerance studies thus far suggest two sites 
of action of corticosteroids in the metabolism of 
calcium, one at the level of bone and the other 
involving renal excretion; possible action at the 
intestinal mucosa cannot be detected by this tech- 
nique. 

A 66-day metabolic balance study on a normal 
control volunteer was conducted to examine the 
metabolic effects of a new synthetic 6-alpha- 
fluorinated corticosteroid. Previous study on this 
project of a patient with active rheumatoid 
arthritis had demonstrated that administration 
of this compound resulted in (a) absence of sig- 
nificant sodium and water retention or of potas- 
sium loss, (b) nitrogen loss as anticipated, (c) 
slight decrease in urinary calcium, and (d) after 
brief initial fecal loss of calcium, progressive 
calcium retention. The normal subject showed 
similar results with respect to nitrogen and elec- 
trolytes but a consistent increase in fecal cal- 
cium. These results, after comparison with 
similar studies by this laboratory with other 
corticosteroids and other patients, both normal 
and with rheumatoid arthritis, suggest that a 
different mode of action of the corticosteroids on 
calcium metabolism exists in patients with rheu- 
matoid arthritis as compared with normal sub- 
jects. Whether this different action has its prin- 
cipal locus in bone or in intestinal mucosa is not 
yet clear. 

Human Total Energy Metabolism 

Obesity. The Metabolic Chamber group is put- 
ting greatest effort into studies of obesity because 
of importance of the latter as a major health 
problem, a problem susceptible in part to attack 
by precise measurements of total energy expen- 
diture. 

The principal question under study, which if 
settled we believe would constitute a significant 
contribution to the very complex matter of hu- 
man obesity, is to establish or disprove the exist- 
ence of a "species" of obese human beings which 
under conditions of normal household activity 
can maintain their body weight at caloric intake 
levels (less than 1,300 calories/day) which would 
be inadequate for most other individuals; this 
state has been termed "hypophagic obesity." 
Prior studies by Olson and others have not in- 



cluded body composition measurements (such as 
fat-free, water-free body mass, sometimes called 
"metabolically active tissue") to provide an ap- 
propriate standard of reference for comparison 
of subjects nor accurate measurement of energy 
expenditure (precision of Metabolic Chamber for 
continuous 24 hour determination ± 100 Kilo- 
calories or less) , nor has caloric balance been fol- 
lowed for periods long enough to be certain that 
weight maintenance on a low caloric intake did 
not represent merely fluid retention, disguising 
the loss of body fat which would provide extra 
calories. Studies being conducted for the Cham- 
ber are rectifying these deficiencies and include 
close observation of extent of physical activity 
and measurement of a variety of biochemical and 
metabolic indices. Active collaboration with Dr. 
Robert Olson, professor of biochemistry, Univer- 
sity of Pittsburgh School of Public Health, is 
planned for the winter of 1961 when he will be- 
gin to make available to us certain patients he 
has characterized as having "hypophagic obesity." 

Current concepts of "metabolic" obesity include 
the possibility that there is a group of individuals 
genetically predisposed to obesity by way of ab- 
normal rates of fat synthesis or mobilization, per- 
haps intensified by physical inactivity. Another 
possibility, it seems to us, is that an originally 
normal individual first develops excess body fat 
through overeating; it then becomes difficult for 
him to lose body fat because obesity may initiate 
a group of influences (physical, physiologic and 
endocrine-metabolic) which promote the mainte- 
nance of positive caloric balance; evidences for 
a number of these influences are accumulating in 
studies of excessively obese subjects. The most 
provocative (preliminary) finding along these 
lines is the pattern of urinary corticosteroid ex- 
cretion which resembles a functional Cushingoid 
state; an abnormal pituitary-adrenal cortical in- 
fluence in obesity would tend to retard fat mobi- 
lization and utilization. 

Current studies of obesity have been directed 
primarily toward study of the relative influence 
of enforced energy expenditure by exercise on 
appetite and on rate of weight loss during caloric 
restriction, and also study of work capacity 
(cardio-pulmonary function during exercise) in 
the obese subject. Studies thus far on four obese 
women and on four women of normal weight 
have indicated the following principal findings 



190 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



among several noted: 1) Exercise clearly con- 
tributed to the daily negative caloric balance on 
intakes of 650 to 1,000 kilocalories per day, as 
demonstrated by 24 hour measurements of energy 
expenditure in the Metabolic Chamber; and 2) 
Energy expenditure at standard treadmill rates 
and slopes decreased progressively as body weight 
was lost by obese subjects; when obese subjects 
were re-loaded with a canvas vest containing lead 
shot to a previous natural weight, energy expen- 
diture per unit weight transported was usually 
less, indicating inefficient transport of obesity 
tissue as compared to inert weight. 

Energy Metabolism in the Cold; Relation to 
Body Fat. A paper is in preparation on a study 
of the energy metabolic response to 50°F. expo- 
sure involving 8 male and 1 female subjects 
ranging in body fat content from 15 to 45 per- 
cent. In summary of the principal findings, a 
specific formula was derived (with the aid of 
Dr. J. Z. Hearon, Office of Mathematical Re- 
search, NTAMD), for the inverse relationship 
between percent body fat and resting heat pro- 
duction (energy expenditure response) per unit 
of body surface area. It was demonstrated 
clearly that when obese subjects are exposed to 
cold, their relatively small metabolic response 
results in a net saving of energy as compared to 
the energy balance situation in a lean individual ; 
this represents a modest but definite influence 
acting toward maintenance of positive caloric 
balance in the obese. Body insulation was found 
to be directly related to the amount of body fat, 
but not completely accounted for by body fat. 
Wide individual differences in metabolic response 
were noted among subjects matched in age, pre- 
vious cold exposure and body fat content. The 
subjective reaction to cold was unrelated to the 
metabolic response ; in other words, well insulated 
obese individuals displaying less metabolic re- 
sponse in calories expended than thin subjects 
either might or might not display as intense a 
subjective discomfort in the cold. Metabolic re- 
sponse in the cold was periodic, occurring in 
cycles having periods of 7 and 18 minutes; the 
18 minute peaks were associated with frank mus- 
cular shivering. Characterization of the meta- 
bolic response to cold with the precision indicated 
would not have been possible without instrumen- 



tation of the sort built into the NIAMD Meta- 
bolic Chamber. 

Blood Diseases 

Initial Stages of Blood Coagulation. During 
the past year, these studies have involved chiefly 
evaluation of the metabolism of antihemophilic 
factor (AHF) and the role of AHF in throm- 
boplastin formation. It is well known that most 
clotting factors are synthesized in the liver be- 
cause various factors become deficient at different 
stages of hepatic dysfunction. However, AHF 
never becomes depressed with liver disease (or 
even following hepatectomy of experimental ani- 
mals), and moreover is not depressed by any 
other disease process. Therefore it has not been 
possible to induce AHF deficiency to assess re- 
generation rates in animals capable of forming 
this substance. 

Opportunity to study acquired AHF deficiency 
was afforded by the finding in three patients of 
an unusual coagulation abnormality which proved 
to be complete deficiency of AHF due to an ab- 
normal component in the gamma globulin frac- 
tion of plasma which specifically inactivates this 
factor. The patients studied were two elderly 
females, both of whom had elevated gamma glo- 
bulin levels, and a middle aged man who had a 
normal gamma globulin level. In each case there 
was no underlying disease and no apparent rea- 
son for development of the abnormal gamma 
globulin. Although the physical chemical prop- 
erties of the gamma globulin are those of a 7S 
antibody and its combination with AHF is stoi- 
chiometric, it does not fix complement with or 
precipitate AHF, and therefore cannot be classi- 
fied as yet as a true antibody. Of particular 
interest was the finding that the anti-AHF in as 
little as 25 ml. of a patient's plasma would pro- 
duce a marked AHF deficiency in a normal hu- 
man being, as well as in laboratory animals. This 
has permitted for the first time an evaluation of 
regeneration rate of AHF in normal individuals 
and animals and provides a means of determining 
the site of synthesis of AHF. 

Another aspect of the work has been experi- 
mental treatment for the acquired disease. Both 
plasmapharesis and exchange transfusions have 
been used, the latter approach having produced 
a remarkable clinical remission. Methods of as- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



191 



saying effects of the inhibitor during initial stages 
of coagulation have been developed and are being 
used to determine the step in which AHF is in- 
volved in formation of thromboplastin. 

Effect of Divalent Ion Chelators on Blood 
Coagulation. These studies have shown that 
calcium not only is an integral part of AHF and 
Factor V, but also is strongly bound to fibrinogen. 
Some years ago it was found that EDTA inter- 
fered with the coagulant properties of fibrinogen, 
and it has generally been assumed that chelation 
of ionic calcium was responsible for this effect. 
Our recent studies employing highly purified 
fibrinogen indicate that fibrinogen contains 5 to 
10 atoms of firmly-bound calcium per molecule, 
and that EDTA forms a complex with the cal- 
cium attached to fibrinogen, rather than effecting 
a reversal of the calcium-fibrinogen complex. 
Studies which are being pursued further with 
C 1 * labeled EDTA promise to provide informa- 
tion concerning the molecular structure of fibrino- 
gen and the active sites involved in its polymeri- 
zation. 

In evaluating the dissociation of Ca-AHF and 
Ca-Factor V complexes, it became necessary to 
determine the equilibrium concentration of Ca + + 
in mixtures containing Mg ++ , protein capable 
of binding Mg and Ca, and the chelator, citrate, 
oxalate or EDTA. Dr. J. Z. Hearon, Office of 
Mathematical Research, NIAMD, has derived 
formulae for calculating equilibrium concentra- 
tions of the divalent cations in these complex 
mixtures. This has permitted a precise compari- 
son of the conditions which reverse calcium clot- 
ting factor complexes, as well as the minimum 
amount of free calcium required for various co- 
agulation reactions. Although these studies were 
undertaken with respect to the coagulation sys- 
tem, evaluation of the concentrations of free ions 
in complex mixtures has bearing on numerous 
physiologic problems involving divalent cations. 

Factor VII. These studies have been continued 
chiefly with respect to developing a better form 
of therapy for the congenital deficiency state and 
determining the survival of the factor in the 
circulation. We have used a concentrate which 
permits administering a therapeutic dose of Fac- 
tor VII in a 2.5 ml. volume instead of the 250 ml. 
f\f plasma previously required. Recent observa- 



tions suggest that the concentrate is adequately 
absorbed following subcutaneous or intramuscu- 
lar administration and that a simply adminis- 
tered prophylactic dose may be entirely effective. 
Ability to provide large amounts of the material 
in a single small injection has permitted separa- 
tion of the rate of exchange with extra vascular 
spaces from the true biological life. 

Proteolytic Enzyme Therapy for Intravascu- 
lar Thrombosis. The euglobulin fraction of 
human plasma contains a precursor substance 
called plasminogen, which on the addition of 
streptokinase (SK), a substance in the culture 
filtrate of streptococci, develops proteolytic ac- 
tivity. SK produces a second activity in human 
plasminogen called "activator" activity, which 
is capable of provoking proteolytic activity in 
plasma of animals which do not contain activator. 
In every step of the purification of human plas- 
minogen, both the precursor of the proteolytic 
enzyme, plasmin, and the precursor of the "acti- 
vator" activity are concentrated equally. It has 
been concluded from most kinetic analyses in the 
literature that plasminogen is a mixture of two 
substances and that streptokinase first activates 
the "activator", then the "activator" produces the 
proteolytic enzyme, plasmin. Our studies of the 
kinetics of this system indicate that plasminogen 
is a single substance which, under the influence 
of SK, forms two consecutive derivatives, the 
first being the active enzyme, plasmin, and the 
second an altered form of plasmin which is cap- 
able of provoking proteolytic activity in animal 
plasma. The steps in the reaction can be differ- 
entiated by specific inhibition of the intermediate 
derivative with soy bean trypsin inhibitor 
(SBTI) : 

SK SK 

Plasminogen >- Plasmin >- Activator 



+ SBTI 



Inactive Complex 

A New Immunologic Disease. A thrombocyto- 
penic state which develops in the absence of any 
apparent underlying disorder represents the rela- 
tively common syndrome of idiopathic thrombo- 
cytopenic purpura (ITP). Similarities between 



192 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



this syndrome and thrombocytopenic states which 
can be produced experimentally with antiplatelet 
antibodies suggest that ITP is caused by an im- 
munologic process. However, up to now neither 
a definite antibody nor a naturally occurring anti- 
gen has been implicated in ITP. Studies done 
in the past year concerned differentiation of a 
specific immunologic type of purpura which 
heretofore was indistinguishable from ITP. The 
pathogenesis of the disease proved to involve 
mismatch of an inherited platelet antigen and 
subsequent development of a complement-fixing 
antiplatelet isoantibody which was capable of 
producing severe thrombocytopenia in the sensi- 
tized individual. Complete and immediate cure 
was produced by exchange transfusion. 

The antigen and antibody involved have been 
purified and extensively characterized, the nature 
of the immuno reactions have been defined pre- 
cisely, and the genetic control of the platelet 
antigen has been fully characterized. 

The following are some of the most interesting 
entirely new facts in the field of immuno-hema- 
tology which were derived from this study: An 
allelic pair of autosomal genes in human beings 
determines three platelet genotypes which can 
be distinguished by quantitative immunologic 
studies as phenotypes. This is the first time 
blood cell phenotypes corresponding to the geno- 
types have been differentiated. An antigen on 
platelets of 98 percent of the local population is 
lacking on platelets of 2 percent. Thus mis- 
match of the antigen occurs in approximately 
2 percent of all transfusions. The antigen fortu- 
nately is only weakly antigenic. Even if an 
antibody forms, it is unable to react with the 
recipient's platelets unless some free antigen re- 
mains in the circulation to coat the recipient's 
platelets and permit attachment of antibody. 
This is the first time that transfer of a free 
cellular antigen has been implicated in a disease 
of sensitivity. Exchange transfusion produced 
a cure primarily by effecting removal of free 
antigen rather than removal of antibody. 

The most sensitive technique for measuring the 
immunoreactions involved is quantitative com- 
plement fixation; for other techniques generally 
applied in immuno-hematology such as platelet 
agglutination, Coombs serum procedures, and 
staining with fluoresceine-tagged antibodies were 
an order of magnitude less sensitive or completely 



useless. Just as in the case of drug antibodies 
which we have studied, the present antibody was 
found to produce thrombocytopenia in vivo in 
man and animals at concentrations which are not 
detectable by the most sensitive techniques avail- 
able. Immunoreactions of the type delineated 
could account for a number of diseases consid- 
ered to be "autoimmune" processes only because 
no foreign antigen has been identified as yet. 

A New Purpuric Disease. In 1955 Gardner and 
Diamond described an unusual form of autosen- 
sitivity in which the patient's own red cells pro- 
duced large painful ecchymoses when extra- 
vasated into the skin. We found that these 
lesions, which were produced by as little as 6 
micrograms of red cell stroma, could be precisely 
duplicated by intradermal injections of as little 
as 1 microgram of histamine or by injection of 
any agent which released skin histamine (such 
as basic amines or trypsin). The conclusions 
were that ecchymoses were mediated by hista- 
mine, released as the result of an antigen-antibody 
reaction occurring intradermally. Since then we 
have had opportunity to study five other patients 
with a similar disorder but with varying sensi- 
tivity both to histamine and to red cell stroma. 
As a result we have defined a spectrum of symp- 
tomology which these patients may present, and 
have thereby shed some light on the nature of 
obscure fixed tissue antibodies and their effects 
on vascular permeability. 

Antibody Reactions in Anemia and Purpura. 
Following our finding that the complex reactions 
which take place between quinidine, antibody, 
platelets, and complement in quinidine thrombo- 
cytopenic purpura are the same as the reactions 
which take place between stibophen, antibody, 
red cells, and complement in stibophen hemolytic 
anemia, we have continued work with the rare 
antibody induced by stibophen in an attempt to 
resolve the question of antigen specificity, meas- 
ure the valence of antibody, and determine the 
number of sites for antibody attachment per cell. 
These studies have required development and 
modification of methods for measuring extremely 
low concentrations of catechols by spectrophoto- 
metric, spectrophotofluorometric, and isotopic la- 
beling techniques combined with immuno-elec- 
trophoresis. Progress so far indicates that the 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



193 



first step of the over-all reaction which results 
in an antibody-drug-cell-complement complex is 
the attachment of drug to antibody. This is an 
important finding, for if the first step of complex 
formation is combination of antibody with drug, 
rather than cell with drug, the implications are 
that the cell-drug complex is not the antigen but 
that the antibody-drug complex may be non- 
specifically adsorbed on cell membranes, just as 
other non-antibody plasma proteins are adsorbed. 
Explicit information concerning this type of im- 
munoreaction and its effects on cells is essential 
in order to provide a basis for interpreting the 
obscure, apparently nonspecific reactions between 
cellular components and gamma globulin in dis- 
eases such as rheumatoid arthritis, Sjogren's syn- 
drome, and lupus erythematosus. 

Pediatric Metabolism Branch 

In the first year of operation, the Pediatric 
Metabolism Branch has devoted most of its efforts 
to the investigation of cystic fibrosis of the pan- 
creas: 51 patients with this disorder have been 
studied. Other diseases leading to malabsorption 
in children and disorders due to glycogen stor- 
age have also been the object of study. 

Cystic Fibrosis of the Pancreas 

Principal aim of the investigations being con- 
ducted on cystic fibrosis is to elucidate and, if 
possible, uncover the basic defect in mucopoly- 
saccharide metabolism presumably responsible for 
the generalized disturbance of exocrine secre- 
tions which gives rise to the clinical manifesta- 
tions of this disease. 

Mucus Structure and Mucopolysaccharide Metab- 
olism, 

Evidence was obtained in previous studies of 
the occurrence in duodenal fluid of patients with 
cystic fibrosis of mucopolysaccharide compounds 
characterized by abnormal chemical structure and 
physico-chemical behavior. These mucoprotein 
fractions have an increased fucose and decreased 
sialic acid content affecting their solubility in 
organic solvents. If this obtains in mucous se- 
cretions throughout the body, it would be possible 
for a change in the physico-chemical environment 
of body fluids to cause irreversible precipitation 
of imicoproteins in structures such as the pan- 



creas, liver, and bronchi, thus initiating the chain 
of clinical events and leading to many of the 
symptoms manifested by patients with this dis- 
ease. 

In collaboration with Dr. Dische, Department 
of Biochemistry, Columbia University, further 
investigations along these lines have been actively 
pursued in duodenal juice, urine, and other secre- 
tions of patients with cystic fibrosis. Work is 
proceeding on isolation and further analysis of 
these mucoprotein components by fractionation 
with organic solvents, continuous-flow electro- 
phoresis, and chromatography. Factors which 
may influence the solubility, physical characteris- 
tics, susceptibility to precipitation and denatura- 
tion of these abnormal mucoid components are 
being tested and preliminary findings have been 
obtained which may be of major importance in 
explaining the genesis of the disease. 

Electrolyte and Genetic Studies 

In cystic fibrosis there is a unique abnormality 
of sweat, saliva and tears leading to marked in- 
crease in the sodium chloride content of these 
secretions. Evidence has been obtained in previ- 
ous studies and in the course of present investi- 
gations that varying genetic endowment may 
affect not only the levels of electrolytes in these 
secretions, but also their response to certain types 
of stress (e.g., dietary salt restriction, administra- 
tion of steroids, environmental heat). The elec- 
trolyte behavior is quite different in this recessive 
disorder in patients with all of the manifestations 
of cystic fibrosis (homozygotes) and those with 
incompletely manifested disease (heterozygotes). 
In addition, from these observations there is 
reason to believe that cases occur in which the 
gene is only partially expressed. Such investiga- 
tions are being pursued by means of metabolic 
studies of sodium chloride metabolism and meas- 
urement of various parameters of adrenal func- 
tion. It is hoped that this approach will lead 
to further clarification of the genetic transmis- 
sion and basic mechanisms involved in cystic 
fibrosis. 

Determination of electrolyte levels in sweat, the 
so-called "sweat test," is the cornerstone of diag- 
nosis in patients with all of the manifestations 
of cystic fibrosis. However, many variable fac- 
tors are involved in these determinations (e.g.: 
rate sweating) impairing the usefulness of this 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



test in family studies and in patients who have 
only some, but not all, of the manifestations of 
the disease. Attempts are being made to perfect 
the methods used so as to eliminate possible er- 
rors and enhance the value of this assay in diag- 
nostic and genetic studies. 

The possible relation of cystic fibrosis in chil- 
dren to many chronic pulmonary and common 
gastrointestinal disorders in adults is one of the 
more challenging leads at the present time. It is 
planned to pursue these investigations actively 
and a beginning has been made in this direction. 
In addition, further definition of other condi- 
tions in the pediatric age group leading to chronic 
lung involvement (e.g., bronchial asthma and 
others) is being attempted. 

Intestinal Absorption 

Evidence has been obtained in previous investi- 
gations by means of intestinal fat and fatty acids 
labeled with I 131 that pancreatic insufficiency may 
not be the only factor involved in the intestinal 
malabsorption in cystic fibrosis. These researches 
are being pursued with the hope of further elu- 
cidation of the mechanisms involved. 

Fat and nitrogen balance studies are also being 
conducted to define the impaired metabolism 
and defective development and nutrition present 
in fibrocystic patients with only partial pancre- 
atic deficiency. In addition, it is planned to in- 
vestigate the effects of varying dosages and 
different kinds of pancreatic extracts on the in- 
testinal absorption of patients with pancreatic 
achylia. 

Pulmonary Involvement 

The pulmonary involvement dominates the 
clinical picture and determines the fate of pa- 
tients with cystic fibrosis. Therapeutic trials of 
antibiotic drugs, of enzymes and physical meth- 



ods of treatment of this complication are being 
actively pursued. Preliminary results have been 
encouraging. 

Intestinal Malabsorption in Children 

Many of the techniques perfected for the study 
of cystic fibrosis can be applied to further in- 
vestigation of other diseases leading to intestinal 
insufficiency in children. The recently developed 
fat absorption tests by means of fats tagged with 
I 131 , the oral small intestinal biopsy, and the 
recognition of wheat and rye gluten as a noxious 
factor in the diet of many pediatric patients with 
malabsorption offer new tools for investigation 
of this very confused field. Several children 
with a variety of conditions leading to this clin- 
ical manifestation have been studied in the past 
year. 

Glycogen Storage Disease 

Congenital and usually familial errors of car- 
bohydrate metabolism lead to a group of dis- 
orders characterized by accumulation of glycogen 
in various tissues and organs leading to enlarge- 
ment and dysfunction of the structure involved, 
and frequently to death. 

In recent times by the application of chemical 
techniques several different diseases have been de- 
fined, each one characterized by the absence of 
an enzyme necessary for completion of the carbo- 
hydrate cycle. Much further definition is neces- 
sary as to the metabolic consequences and clin- 
ical manifestations of these conditions. This rep- 
resents a very dynamic field and other syndromes 
will undoubtedly be recognized as investigations 
are pursued. 

A beginning has been made in this direction 
and several patients with disorders of glycogen 
storage have been studied. 






NATIONAL INSTITUTE OF MENTAL HEALTH 



BASIC RESEARCH 



Introduction 1 



This is my fourth and final annual report. 
Since each of the preceding reports undertook a 
discussion of general questions and since the 
questions themselves are interrelated, it may be 
helpful to introduce this last report by a resume 
of major points considered. 



The first report, written in 1957, depicts the 
urgency for research which will enable men to 
understand how to substitute a just system of 
law for the present international anarchy. The 
most staggering problems facing man are no 
longer those of food, clothing, shelter, power and 
transportation, although these are not yet equably 
distributed; instead, the problems he knows least 
how to solve and which cause him the greatest 
tension are those relating to the perception of 
actions and of shibboleths, the translation of 
ideas, the momentum of traditional concepts, the 
adhesive behavior of groups, and the communi- 
cation of ideals and goals. Men of all nations 
are in need, promptly, to possess vastly improved 
means for comprehending and coping with a 
myriad of problems relating to perception, mem- 
ory and emotion. Men need urgently to learn 
how to become more constructively adaptive as 
interdependent individuals within a completely 
comprehensive, planetary level of social integra- 
tion. 



i Written by Robert Livingston, M.D. 

As in previous annual reports, the laboratory chiefs have pro- 
vided comprehensive statements of research progress achieved 
during the year. I am attempting here to continue an explora- 
tion of questions relating to broader horizons of the mental and 
neurological sciences. In the Immediacy and seeming urgency 
of our daily existence, we tend to put these questions aside as 
if they were not prime to our purpose. Yet, I believe that they 
are truly pertinent to our ultimate best contributions to science 
and to our fellow man. These annual reports ought to be con- 
sidered as fragments in a continuing insight-seeking discourse 
rather than as pretensions of discovered truth. 



Several research domains relating to brain and 
behavior are pertinent to the solution of the so- 
cial and political problems that now seem so 
overwhelming. We need urgently to know how 
to increase man's capabilities for adaptation to 
an environment wherein his greatest threat arises 
from other men. Man's aspirations for rational 
action, and his definitions of what is rational, 
depend in large measure upon insights which 
can be gained only through an improved under- 
standing of the fundamental processes of brain 
and behavior. Ignorance of the limits and poten- 
tialities of these processes acts as a handicap in 
present attempts to solve our most pressing so- 
cial and political problems and leaves man's adap- 
tive behavior almost exclusively subject to brutish 
prescientific impulse. 

Defense and diplomacy cannot be primarily de- 
voted to the advancement of concepts underlying 
interpersonal and social integration: they must 
proceed on the basis of whatever is the con- 
temporary level of understanding of human rela- 
tions, no matter how inadequate this may seem 
in the context of their problems. Defense and 
diplomacy are, nevertheless, providing a precious 
gap of time within which the essential political 
and social growth and adaptation must take 
place. It is what we accomplish during this gap 
of time by way of advancing knowledge and en- 
couraging constructive social adaptation that will 
count effectively. Progress in the discovery and 
communication of more rational ways to improve 
human relations will itself act as a relaxant to 
threatening tensions. Progress will be increas- 
ingly rapid as we deliberately cultivate the best 
opportunities for thoughtful and creative re- 
search enterprise in these fields. Our view is that 
the brain-behavior system is an evolutionary 
product which is playing its role in human devel- 
opment just as do teeth and claws : yet from this 
great transactional organ we can expect far more 
constructive and creative potentialities than from 
teeth and claws. The brain-behavior system, in 



195 



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ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



our view, is a very incompletely exploited instru- 
ment for survival. 

A further theme of the 1957 report discusses 
the essential role of creativity — the sine qua non 
for conceptual progress in any field. No new 
level of understanding can be achieved without: 
(i) a substantial mastery of the field of in- 
tended accomplishment; (ii) an internal dedica- 
tion and discipline devoted to a program of 
thought and action which has to surpass the con- 
temporary limits of conception; (iii) a capacity 
for nimble imaginative interplay of ideas and 
images; (iv) a confident degree of intellectual 
nonconformity; and (v) a capacity to communi- 
cate effectively the newly acquired conceptual 
formulations. It is obvious that these criteria 
place special demands not only upon individuals 
who aspire to be scientifically creative, but also 
upon institutions which aspire to recruit and re- 
tain creative scientists. 



II 



The second in this series of Annual Reports, 
that for 1958, substantiates that all branches of 
science, and indeed all learning, depend in their 
ultimate formulations upon concepts relating to 
human sensory, mnemonic, judgmental and effec- 
tual mechanisms. Our present concepts relating 
to these functions are not fixed and immutable 
but are changing rapidly. As insight is extended 
we shall be able to improve the operational defi- 
nitions of reality relating to all branches of 
learning, those concerned with the rest of the uni- 
verse as well as with ourselves. Our profoundest 
concern as scientists is with the intellectual con- 
tent of our disciplines and with the creative proc- 
esses essential to achieving substantial progress, 
as contrasted to fashionable satisfaction, through- 
out these fields. All administrative considera- 
tions should be devoted to the development, en- 
couragement and exercise of these essentially 
intellectual faculties. 

A further point of the 1958 report is that the 
idea of the separateness and incommensurability 
of mind and body is not an automatically self- 
understood feature of human existence. Such a 
division is not presupposed by other thoughtful 
■cultures. Our contemporary dualistic assumption 



is inherited directly from Greek scholars who first 
entertained this idea at a particular moment in 
history, around 400 B.C. No notion of the sep- 
arateness of mind and body appears before that 
time. However, western civilization has sustained 
the idea for such a long time that it has become 
deeply imbedded in our language and habits of 
thought. We escape from the idea only by cir- 
cumlocutions and we seldom really abandon it. 
I believe that this assumption is not only unnec- 
essary but that it acts as an obstacle to our 
search for a more adequate understanding and to 
our efforts in the prevention and cure of mental 
and neurological disorders. This handicap is 
also to be regretted because dualism fosters pro- 
fessional, intellectual and conceptual isolation 
among scientists who are trying to understand 
whole man. 

Ill 

The 1959 annual report concerns relations be- 
tween science and society. Science as a human 
value, the place of science in the evolution of 
human values, the contribution of science in ra- 
tionalizing and liberalizing society; the require- 
ment in science that professional responsibility 
should pervade all scientific activity as well as the 
relations between science and society; the require- 
ment for evaluating scientists and for evaluating 
science as a body of knowledge, and for making 
decisions which commit a large proportion of so- 
ciety's wealth to scientific enterprise; the prob- 
lems generated by the cultural interface between 
science and Government, and the administrative 
consequences of these considerations, are delib- 
erated. 

The balance of the third report describes bio- 
logical and social evolution as these proceed to- 
ward greater degrees of obligate cooperation and 
interdependence. Each step is achieved at the 
expense of more arbitrary modes of behavior 
which operate at lower levels of biological and 
social integration. This loss is counterbalanced 
by substantial gains in freedom and power for 
self-determination enjoyed by successively higher 
levels of organizational integration. The pro- 
gressive enlargement of freedom within the more 
highly integrated systems can best be appreci- 
ated by scanning biological and social evolution 



NATIONAL INSTITUTE OF MENTAL HEALTH 



197 



over a longer time scale than is usually con- 
sidered for the next essential step, that is, the 
establishment of a just and adaptive system for 
international integration. From these consid- 
erations follow certain implications relating to 
the comportment of the individual, family com- 
munity, institution, nation and international or- 
ganization in the direction of providing a scheme 
for social integration on a planetary scale. 

IV 

In this final annual report my purpose is to 
examine some of the biological limitations and 
opportunities which underlie human capabilities 
for the social and cultural adaptations essen- 
tial to survival. First, it is necessary to ex- 
amine three challenging problems. 

"* * * we are here as on a darkling plain 
Swept with confused alarms of struggle and 

flight, 
Where ignorant armies clash by night." 

Matthew Arnold 

"War is the chief stupidity of man" 

Charles Erskine Scott Wood 

The Technological Challenge 

In what significant ways has technology altered 
individual and national powers for action? In 
what ways does this automatically narrow the 
focus of responsibility for perceptions and judg- 
ments conditioning the utilization of national 
powers ? 

Within recent years it has become possible to 
pulverize the largest cities with only two or three 
packages of explosive. All other cities can be 
destroyed with a single package. There are 
many simple as well as sophisticated ways of 
delivering these packages. The number of pack- 
ages available is said to be greater than the 
number of civic targets. Policies of threat and 
retaliation among the most powerful nations are 
roughly balanced, in an uneasy equilibrium, 
through the competitive development and de- 
ployment of instruments for massive extermina- 
tion. For the first time mankind as a whole 
is vulnerable. The world is now a decade and 
a half deep into a gigantic arms race. History 
warns us that no previous arms race has ever 
ended peaceably. 



Packages of modern explosive can be dis- 
patched on the initiative of only a few men. 
They can be sent off from a variety of loca- 
tions and delivered to any civilized target on the 
globe within minutes. Since surprise assault 
might blunt or disorganize counterattack, each 
antagonist is obliged to maintain himself in 
readiness for momentary massive retaliation. 
Within the last half century, the interval for all- 
out commitment relative to national security has 
shrunken from months to minutes. Deliberate 
judgment and widespread consultation must take 
place well in advance of the critical moment and 
must be based on forecasted probabilities and 
contingencies which are impossible to anticipate 
completely. 

Several non-atomic powers are busily prepar- 
ing themselves for their own independent na- 
tional capability for massive extermination. 
There will be several more members and mem- 
bers-by-alliance in the "nuclear club" within 5 
years. Just what extremity of desperation in 
any given nation will trip off an international 
struggle involving massive extermination is no- 
where publicly defined. In several countries, na- 
tional purposes and national desperation are 
rather vaingloriously and personally defined. A 
large number of existing and potential triggers 
are capable of frustrating national purposes and 
of aggravating desperation. These triggers may 
suddenly reinforce one another in an uncontrol- 
lable "avalanche effect." These triggers are not 
under the confident control of any individual, any 
nation, or any existing combination of nations. 

National constraints against the use of weap- 
ons for massive extermination may be moral or 
constitutional in character or based on fear of 
retaliation. They may also be based on the reali- 
zation that many untried and possibly more suc- 
cessful ways to further national purposes exist 
and might be explored. There are no enforce- 
able constraints operating at the international 
level. Until constitutional machinery is adopted 
which will ensure an acceptable system for in- 
ternational justice, each nation is left to its own 
dreadful devices. Even if all of the present 
international crises were settled, other alternative 
and emergent problems would become equiva- 
lently critical. A quick riffle through the chron- 
icles of history will illustrate that men have to 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



face a continuing challenge of getting along with 
one another and of sharing the world's resources. 

Although men bemoan the cost of weapons for 
massive extermination, the cost itself does not 
provide notable constraint : weapons for massive 
extermination yield an order of magnitude less 
expensive destruction than any previous military 
techniques. The costs seem extraordinarily high 
only because the world is preparing the means 
for many orders of magnitude more destruction 
than was ever before contemplated. If general 
destruction is man's principal objective, he is 
now pursuing full tilt the most efficient and 
economical courses by which to achieve that end. 

Communication of threatening events and 
polarization of national attitudes is rapid and 
world-wide. Unfortunately, it is commonplace 
now to suppose that charges and insults must be 
answered practically instantly and in the same 
vein ; any other reaction is apt to be misconstrued 
at home and abroad. 

All of this has vastly changed the nature and 
potentialities of individuals and nations and is 
altering the whole fabric of human relations. It 
has placed the fate of all mankind and the dis- 
posal of all the monuments of human achieve- 
ment at the disposal of a relatively few persons 
scattered around the world who have in their 
control practically unlimited powers for destruc- 
tive action. The situation in which these men 
must operate on behalf of the national complexes 
they represent is dynamic and worsening. Most 
importantly, the individuals concerned are them- 
selves committed in percepts, passions and judg- 
ments according to their own life experience 
which, of course, has largely been dedicated to a 
leadership role in their own idiosyncratic society. 
We have not yet comprehended, much less be- 
gun to treat rationally, the vast implications 
stemming from this technological revolution. 

The Social Challenge 

Unbelievable force has come into man's posses- 
sion just as he has begun to find out that dis- 
tortions deeply affecting perceptions, feelings and 
judgments are normally and inevitably incorpo- 
rated as part of the baggage of all adults. 
World-wide communication has become practi- 
cally instantaneous just as man has begun to 
appreciate the biological crudeness and inade- 
quacy of the transactions of language. The world 



is becoming overcrowded just as man is begin- 
ning to appreciate the social value of the indi- 
vidual and beginning to develop a primitive in- 
sight into constructive human relations. Man 
has become proficient at polarizing mass attitudes 
of what is right and what is wrong, but is 
scarcely as far along in fundamental ethical rea- 
soning (if, indeed, as far) as some of the ancient 
Greeks. There is as yet too little cognizance of 
the range of assumptions and criteria upon which 
every system of rectitude is based, and too little 
patience with the requirements for cross-cultural 
understanding. 

Man still faces the same two old choices if he 
would avoid war: either he must discover and 
establish institutions of government that will 
substitute a universality of values, purposes and 
means for the present conflicting systems, or he 
must provide institutions that will admit and 
protect differences of values, purposes and means, 
but yet guarantee agreement in those actions es- 
sential to maintain order and at the same time 
accommodate change. As Walter Millis has de- 
clared of a world without war: "it would still 
be true, in the sense in which Jefferson pre- 
sumably meant it, that 'the tree of liberty' would 
have to be 'watered by the blood of martyrs.' 
Men would still die for principle or passion ; but 
they would not die as the helpless, systemic vic- 
tims of a world order based upon highly organ- 
ized and armed national sovereignties. Deprived 
of the easy simplicities and illusory securities of 
the war system, statesmanship would meet more, 
not less, difficult problems than those it must now 
confront; and it would take brains, illuminated 
by vision in the leaders and education in their 
followers to surmount them * * * A world from 
which "organized war has been excluded would 
not be an easy one, and it would raise threats 
to various groups, economic interests, ideals and 
convictions which may well seem greater than 
the (still almost unimaginable) threat presented 
to all by a continuance of the war system itself. 
But it would be a viable world * * * its attain- 
ment, while immeasurably difficult, does not seem 
to be impossible." 

We have come only a little way in the required 
direction at the individual, national and interna- 
tional levels. For instance, the entire budget, 
from all member nations, for operations of the 
United Nations, the only organization having an 



NATIONAL INSTITUTE OF MENTAL HEALTH 



199 



internationally endorsed charter for arranging 
the settlement of international disputes, just 
equals the size of the budget for cleaning the 
streets in New York City. The cost of the 
United Nations, prorated over the world popula- 
tion, is less than two cents annually. The yearly 
cost for armaments alone, not including espio- 
nage, propaganda and conventional diplomacy, 
prorated in the same way, is more than three 
thousand times greater than this, and uses up 
nearly half of the total product of human and 
other resources on a world scale. 

1. The need for purposeful aspirations on a 
world scale. The most critical weakness of hu- 
man society today is its lack of concerted purpose 
to achieve more comprehensive social and politi- 
cal integration. It is widely acknowledged that 
men of all nations are thrown together perforce 
of modern technology, but the consequences of 
this fact are not sufficiently taken into account 
in men's actions. It is as though we really be- 
lieved that if we stood upon our traditions, the 
present state of the world would revert to some 
more comfortable epoch of the past. Such faith 
is folly. 

Every human shares certain fundamental in- 
terests and objectives with all others: that it is 
better to be alive than dead, fed than starved, 
free than enslaved. There are other shared inter- 
ests, but none is more fundamental and none is 
in greater present jeopardy. Existing social sys- 
tems are not arranged so as to safeguard even 
these rudimentary needs on a universal scale. 
It is rare for any existing system to advocate 
their fulfillment for anyone who does not belong 
to the same system. No individual can safeguard 
fulfillment of these needs for himself, nor for 
any other. No social system can safeguard them 
for its own people, nor for any other — although 
we continue to extend our faith in this possi- 
bility. The chief problem is to establish a new 
system that is sufficient to satisfy the needs for 
survival, sustenance and self-realization for all 
mankind. Of course, people will not then be 
satisfied, for even though alive, fed and free, they 
will still have problems enough; there will re- 
main plenty of difficulties to challenge man's 
capacities for further social progress. 

2. The need for purposeful actions on a world 
scale. The dimensions of social need and the 
arena of struggle for power and wealth are 



world-wide in scale. Perforce of these facts, 
creative thinking of ways to satisfy fundamental 
human needs and at the same time to arrange 
for the just settlement of disputes must be con- 
ducted on a global scale. There is now no way 
to safeguard our lives or our welfare without 
achieving such safeguards for everyone. We 
need a fresh analysis of the role of all institutions 
bearing directly or indirectly on the requirement 
for a world government that supports universal 
maximum individual self-realization. Whether 
traditional ideas and traditional institutions can 
be helpful should not be presupposed prior to a 
thorough, objective and disinterested analysis of 
the requirement. What was adequate for less 
extended needs and aspirations will probably not 
now suffice. Paralleling this, we need research 
to enlarge man's grasp of the processes of human 
relations. Analysis, research and the required 
revision of institutions should be carried out with 
the deliberative participation of as many as pos- 
sible of those concerned, that is, with the widest 
possible world base. This is essential in order to 
enable the consideration of this problem among 
the world's most creative thinkers, of which no 
single nation has a monopoly; moreover, world- 
wide participation will ensure insight into the 
requirements and means for modifying all perti- 
nent institutions in the desired direction. 

No nation or confederation of nations, acting 
unilaterally, can put world government into ef- 
fect. This cannot be accomplished either by 
suasion or coercion because any unilateral en- 
deavor giving evidence of the likelihood of suc- 
cess would undoubtedly provoke a full scale war 
before it had gotten very far. Thus, the only 
alternative to the present uneasy situation seems 
to be to invite the broadest world base of creative 
social thinkers to undertake research and deliber- 
ations leading to the development of plans for 
a more inclusive community of mankind. This 
undertaking of research and discussion may not 
yield ideas upon which agreement can be estab- 
lished in the near future; but, if this objective 
is purposefully undertaken, sound and satisfying 
ideas are ultimately achievable. 

We have substantial reason to hope for prog- 
ress in this enterprise through application of the 
methods of science. Social evolution in the past 
has come about through far slower and more hap- 
hazard processes. When research and discussion 



200 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



dedicated to this goal get underway, this fact by 
itself will encourage world leaders and mankind 
as a whole to be more tentative, to tolerate greater 
ambiguities and to exercise more patience con- 
cerning international relations. The findings of 
such research and the results of world-based de- 
liberations as to its application can be submitted 
for national recognition or rejection; popular 
referenda might be appropriate for the determi- 
nation of final acceptability of the evolved plans. 

The world prospect will be improved by the 
encouragement and development of a world-view 
in these undertakings; not a view to conquering 
the world but to making it safer and, in other 
basic ways, better for all mankind. Naturally, 
attitudes and impulses which during the last four 
centuries have worked to create strong nation- 
states have established a prominent place in men's 
thinking and ways of life. Our United States' 
national experience of nearly two centuries and 
our individual strivings to improve national 
greatness, national security and national welfare 
are dominant features of our own existence. 
These attitudes and impulses should not be dis- 
carded in assuming a world-view. A world-view 
and world-government appear to be the only pos- 
sible ways for securing enduring national great- 
ness, national security and national welfare. 
Greatness, security and welfare cannot be sus- 
tained indefinitely on a merely national scale. 
As a nation, we are beginning to recognize this 
and to put it into increasing practice in our for- 
eign and defense policies, foreign aid, military 
aid and mutual security programs. Something 
new has already been added to nationalism, some- 
thing which obviously accommodates continuing 
patriotic devotion to nationality. We are learn- 
ing to be more rational about more complex and 
more inclusive human transactions, to perceive 
the consequences of our actions in a wider context 
and in a longer view. Growth in freedom and 
opportunity for self-realization is known to in- 
crease with each more inclusive level of social 
integration and will increase even more when 
our identifications are extended to include all 
mankind. 

3. Urgency of action. Most leaders in the 
powerful nations appear to comprehend that pre- 
cipitation of all out war amounts to mutual 
murder and suicide: at present, a metastable 
military stalemate exists. This metastable stale- 



mate, however, can be easily upset by desperate 
individuals and groups, even among the least 
powerful nations. They may conceive they have 
nothing to lose and perhaps something to gain, 
or they may simply behave irrationally. There- 
fore, time is our most precious commodity. We 
need to proceed in all deliberate haste toward the 
goal of world-wide social integration. What 
kind of time schedule might be estimated for 
this task? The necessary research and discus- 
sion need time; too urgent a desire to arrive 
quickly at an agreed upon plan may lead to in- 
adequate improvisation and to the blunting of 
creative efforts which might otherwise provide a 
far more desirable solution. The social and atti- 
tudinal changes required are probably equivalent 
in revolutionary dimension to those which have 
taken place around the world during the last 
fifteen years. Perhaps fifteen years may be ac- 
cepted as a minimum time if we begin immedi- 
ately in a purposeful and dedicated way. On 
the other hand, we cannot afford to wait until 
the beginning of the next century before we 
belong in a true sense to one world. 

4. The "idealistic" course is the only realistic 
course. Mankind can look back with justifiable 
pride on a number of now widely accepted revo- 
lutions in attitude and behavior which grew out 
of enlarged respect for human needs viewed in- 
creasingly objectively. We can expect traditional 
forms of thinking to reject and probably actively 
oppose research and discussion dedicated to the 
establishment of a system for the integration of 
mankind as a whole. Such pursuits may be 
thought of as "visionary," "idealistic" and "un- 
realistic." Yet, if any lesson can be clearly 
drawn from recent history, it is this: that many 
ideas labeled only recently as visionary, idealistic 
and unrealistic have already become the only 
tenable, practical and realistic ones. Much of 
the sinew and solvent of the most powerful na- 
tions of the world has been expanded in the 
process of learning this lesson. 

Ideas, not things, rule mankind. Traditional 
ideas and consistent forms of behavior preserve 
continuity. They are reassuring to our immedi- 
ate associates and bring us such comfort as at- 
taches to the familiar. Yet, in the long run, this 
comfort may be illusory. For centuries, religious 
and racial tolerance were considered by all right- 
minded people to be contrary to any reasonable 



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conception of morality. Epileptics and the in- 
sane were beaten and brutally incarcerated. The 
object of such treatment was to punish and drive 
out evil spirits possessing these individuals. All 
right-thinking people were convinced that this 
was in the best interests of both the victims and 
society. The use of lightning-rods was vehe- 
mently condemned as "an impious attempt to 
defeat the will of God" and as a means of "help- 
ing criminals to escape." Vaccination and even 
anesthesia were abhorred on moral grounds as 
contrary to nature. 

It may bring us comfort that the only period 
of history we have to face is our own. Yet it 
remains to be seen whether we can meet the main 
requirement of our day, namely, to establish some 
kind of world order under a just system of law. 
It is now high time that we assume the obliga- 
tion that our conscious awareness and our con- 
science thrust upon us. It remains to be seen 
whether we can generate the sense of confidence 
and purpose necessary to meet this social chal- 
lenge. If we make our best effort and yet fail 
in this attempt, we cannot lose anything not 
already lost. 

The Individual Challenge 

1. The problem of individual commitment. A 
newborn babe can grow up to achieve a practi- 
cally complete behavioral adaptation to any cul- 
ture regardless of his ancestral background. 
Physiognomic differences scarcely interfere with 
this adaptation. A baby is readily accepted be- 
cause he is commitable, yet uncommitted. Within 
a few years, however, a child's potentialities for 
adaptation to another culture become pro- 
gressively limited. By the time he reaches man- 
hood, indistinguishable behavioral adaptation is 
virtually impossible. Commitment involves in- 
ternal polarization with respect to all aspects of 
life, likes and dislikes, even including things 
which are presumably detached from personal 
involvement. The presence or absence, nearness 
or distance of mountains, flatlands, rivers, and 
the sea come to loom importantly along with per- 
sonal associations in commitment and is notable 
in the commitment of "home-sickness." 

Once the process of commitment has begun, 
in reference to language, customs and habitat, 
everything that is consistent with the initial pat- 
tern is easily and congenially adopted. What- 



ever is contrary to that pattern may be over- 
looked (not even sensed), may be sensed but 
dismissed as meaningless, may be met with sur- 
prise and incredulity, or may be subjectively 
distorted and inappropriately acted upon, as if 
it actually conformed to the familiar pattern. 
The recognition of discordance with previous ex- 
perience is characteristically associated with emo- 
tional excitement — possibly anticipating the po- 
tentialities for threat and opportunity which 
accompany every novel experience. Adaptation 
to new patterns of language, customs and habi- 
tat is a slow and demanding process associated 
with many inappropriate responses. 

Educational processes and the methods of sci- 
ence legitimize and encourage the systematic 
examination and objectification of percepts and 
ideas that may have only the most tenuous cre- 
dentials. The foundations of intellectual growth 
depend upon this cultural legitimization. Edu- 
cation is properly called "the servant of all our 
purposes" (John W. Gardner). Education and 
discovery are associated with the intellectually 
satisfying response of "perspective dissolving 
surprise" (William Gorman). Gradually, un- 
familiar patterns become accepted and ultimately 
incorporated into an automatically functioning 
system of percepts, judgments and actions. Com- 
mon sense is the residue of prejudices acquired 
during education (Albert Einstein). Our ca- 
pacity to perceive, accept and accommodate nov- 
elty slows downs with advancing years — as 
though we become increasingly impounded by 
our commitment. The important thing for us 
to comprehend is that the process of becoming 
committed is an indispensable and central part 
of our biological heritage, that it virtually or- 
dains throughout all our development, recogni- 
tion and reaction to all aspects of life. 

Our biological endowment thus equips us to 
adapt so as to behave "successfully" in a given 
environment. The very factors which establish 
an easy and automatic behavior in our accus- 
tomed environment will operate to our disadvan- 
tage when we are exposed to a differing environ- 
ment. This is especially compelling if we are 
unable to recognize the extent and significance 
of the novelty and deal with it as if it were 
familiar. Adaptation to experience carries with 
it a dominating expectation that the environment 
will be enduringly consistent. In a later section 



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we will examine the extent to which all our sen- 
sations and ideas are dependent — not upon the 
external events we are contemplating but upon 
the purposeful direction our lives have taken as 
a result of our individual idiosyncratic experi- 
ence. An incorporated pattern does not give way 
easily to conversion, even when the necessity for 
a different adaptation is fully accepted intellec- 
tually and is reckoned to be life saving. For 
example, obeying the simple but contrary rule 
of turning your head to the right instead of to 
the left in order to avoid oncoming traffic turns 
out to be no trivial adjustment. Taken from the 
point of view of the need mankind now dimly 
discerns, to comprehend the percepts and judg- 
ments and to anticipate the actions of people 
from entirely different cultures, the problem of 
idiosyncratic commitment can be seen to be inter- 
fering in a pervasive way with rational interna- 
tional behavior. 

2. The problem of conformity. Any kind of 
behavioral adaptation, any kind of individual or 
social progress requires nonconformity: noncon- 
formity with one's own past adaptive, experi- 
ence-guided behavior or nonconformity with the 
adaptive, experience-guided behavior of the 
group. The whole history of intellectual prog- 
ress consists of steps taken which liberate us 
from purely reflexive behavior, from the limita- 
tions of immediate and imperious perception, 
judgment and response. The "inertia" of men's 
minds is reflected in the intellectual turmoil which 
usually attends a shift from one level of under- 
standing to the next: that the world is round, 
that the earth revolves around the sun, that 
blood flows through our vessels "as it were, in 
a circle," that men belong in biological conti- 
nuity with the rest of the animal kingdom, and 
that something as small as germs can fell a 
strong man. Most of these intellectual conver- 
sions took decades and sometimes centuries. Each 
step, small or large, demands a break with the 
consistency of previous thought. Each contrib- 
utor to intellectual progress must be, by defini- 
tion, a nonconformist. As Ben Shahn wrote, 
"Without nonconformity we would have had no 
Bill of Rights nor Magna Carta, no public edu- 
cation system, no nation upon this continent, no 
continent, no science at all, no philosophy, and 
considerably fewer religions. All this is pretty 
obvious. But it seems to be less obvious that to 



create anything at all in any field, and especially 
anything of outstanding worth, requires noncon- 
formity, or a want of satisfaction with things 
as they are. The creative person — the noncon- 
formist — may be in profound disagreement with 
the present way of things, or he may simply wish 
to add his views, to render a personal account 
of matters * * * 

"Yet, when it comes to the matter of just what 
kind of nonconformity shall be encouraged, liber- 
ality of view recedes. There seems to be no 
exact place where nonconformity can be fitted 
in: it must not be admitted into the university 
curriculum — that would produce chaos. In poli- 
tics it is certainly inadvisable — at least for the 
time being. It cannot be practiced in journalism 
* * * In science — least of all, alas !" Shahn goes 
on to conclude that "The degree of nonconformity 
present — and tolerated — in a society might be 
looked upon as a symptom of its state of health." 

Saltatory advancement of social concepts — the 
introduction of ways of thinking that change the 
entire character and direction of social progress, 
that lead to the acceptance of a substantially 
more fundamental ideal, that creatively refor- 
mulate the nature of a social problem or its solu- 
tion, that cut short years of social strife — are not 
likely to occur except where circumstances are 
especially favorable for nonconformist social 
thinking. The concept that "all men are created 
equal" was a product of such circumstances. Im- 
provement in social theory, increase in grasp of 
social problems and their solution depend upon 
advances of this sort — intellectually creative ad- 
vances — and upon their institutionalization. As 
Hastings Rashdall said: "Ideals become great 
historic forces by embodying themselves in insti- 
tutions." 

3. The power of purpose. It is not an accident 
that the words curiosity and cure come from the 
same origin. They imply being full of care, 
taking pains; having a desire to learn about, to 
comprehend; possessing empathy for and, thus, 
striving fully to understand the object of con- 
templation. Our guide for human progress, in- 
dividually and collectively, grows out of our 
humane interests, understood objectively, in the 
long range and in the largest sense, humane 
interests made as consciously self-aware as pos- 
sible. It is not likely that man can attain what 
he does not strive for. And it is impossible for 






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203 



him to seek what he conceives as unattainable. 
Thus far, human purpose has predominantly 
sought provincial advantage. This has been 
gradually enlarged from individual to family, to 
community, to nation, without there being un- 
endurable losses along the way. Provincial ad- 
vantage is now possible only on a world-scale. 

By far the most important influence on social 
progress is consciously directed purpose. Men 
need to instill in themselves a sense of great pur- 
pose and high resolve to gain a more comprehen- 
sive level of social integration. The phrase "that 
all men are created equal" once played a role of 
great importance in the establishment of our 
nation. "That all men are created equal" became 
the reiterated definition of purpose of the Fed- 
eral Government in the campaign utterances of 
Abraham Lincoln. This phrase became Lincoln's 
principal personal and administrative theme and 
his main means for mobilizing what ultimately 
saved the Union. At that time, there was little 
the United States could do, nor was there so 
much at stake for the rest of mankind, regarding 
the global inclusiveness of that phrase. "That 
all men are created equal" has, to a regrettable 
degree, remained rhetorical even within our own 
borders since then. But there is every indication 
that the Founding Fathers took this principle 
seriously; they purposefully established institu- 
tions of government dedicated to its recognition 
and fulfillment. It is our turn now to support 
and improve such institutions and to extend their 
application as widely as possible. 

Education is a servant of our purposes, and so 
is science. Both seek the truth. Both debunk 
authority. They teach that the individual, com- 
munity, nation and the universe are always in 
the process of becoming, and that no part of these 
great transactions can be made to stand still. 
There is a powerful anti-inertial effect on society 
contributed by education and science. Cultural 
integrity and the safeguards formerly dependent 
upon the preservation of traditional patterns of 
human relations can now be guaranteed through 
the broadening experience of education combined 
with the creative contributions of science. They 
emphasize the intrinsically creative aspects of 
man's individual life and his ineluctable capacity 
to develop increasing degrees of freedom for 
himself and his fellow men. 

Education and science are not automatically 



liberalizing. They can be encouraged to seek 
truths of differential value favoring one indi- 
vidual, one company or one nation. New knowl- 
edge can be deliberately fostered and exploited 
to yield powerfully disadvantageous conse- 
quences to other groups. In such efforts, educa- 
tion and science quickly outstrip any other sys- 
tem to achieve unilateral advantage. But the 
same endeavor can be extended to seek truths 
of advantage to mankind as a whole. It is 
reasonable to anticipate that science and educa- 
tion can outperform any other mode of approach 
toward this goal. The community of teachers 
and scientists has been international in charac- 
ter since long before nationalism began to be a 
force in human affairs. Educators and scien- 
tists are professionally trained to be adaptive; 
they are professionally dedicated to the discovery 
of larger and simpler patterns of transaction and 
organization. 

Mainly, it is to facilitate the establishment of 
worthier and more humane purposes that I urge 
the fostering of the broadest horizons in the 
labors of educators and scientists. Mankind has 
amply illustrated that it can always retreat to 
more primitive perceptions, judgments and ac- 
tions. When research, encouraged in this way, 
adds new concepts to the traditional ones, men 
will at least have an enlarged freedom of choice 
and probably they will also enjoy greatly in- 
creased powers for constructive action. The rate 
at which meaningful new knowledge is appear- 
ing in the biological, sociological and psycho- 
logical sciences, and the nature of the new in- 
formation provide confidence that substantial 
progress can be made in the direction of helping 
all men become more constructively adaptive as 
members of interdependent social systems. 

The social sciences have been comparatively 
slow to develop partly because the subject matter 
is vastly more complicated than anything dealt 
with in biology or physics, and partly because 
there has always been a strong tabu against in- 
vestigating social processes. The social sciences 
are now in transition from empirical to theo- 
retical bases. By analogy from other branches 
of science it is known that this kind of transi- 
tion increases tremendously the scope and power 
of such studies. The capacity of the social sci- 
ences to provide insight and mechanisms for 
internationalization of the control of social be- 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



havior will ultimately dwarf the practical con- 
tributions of any other domain of science. The 
most impressive utilitarian value of science will 
attach to science when it becomes effective in 
the shaping of constructive human behavior. 
This contribution will have an equally important 
impact upon the mainstream of human intellec- 
tual activity, the philosophy of thought, vitality 
of ideas, and the generation of security as well 
as welfare for all mankind. The question is not 
whether such achievements are feasible but 
whether they can be instituted within the gap of 
time that may be available. 

The Natural Foundations for Human 
Adaptability 

What may be the bases for our commitment to 
earlier developed views of reality, patterns of 
ideation and judgment, and modes of behavior? 
How and to what extent may these interfere with 
re-adaptability ? How compelling is previous ex- 
perience in shaping and limiting what we per- 
ceive? How compelling is previous experience in 
our interpretation and reaction to novelty? To 
what extent do we remain blind to the uncon- 
scious influences of previous experience? What 
can be done in a positive way toward gaining a 
more adequate insight into both the limits and 
opportunities of human adaptability? 

In this section I shall attempt to be more 
explicit concerning the internal processes by 
means of which man adapts to a given environ- 
ment and by means of which he must re-adapt to 
previously unexperienced patterns. For illus- 
trations, I have selected a number of observa- 
tions for brief outline: many of these are old 
and well substantiated findings of research ; some 
are drawn from commonplace experiences; a few 
are new and must be admitted only tentatively. 
What I seek is a working conceptualization of 
man as an adaptative social being. 

1. Developmental considerations. The pres- 
ence of developing muscle cells in the body of 
the vertebrate embryo induces the specialization 
and maturation of motor cells in brain stem and 
spinal cord. Motor nerves send connections to 
these muscle cells and induce muscular contrac- 
tions before the sensory nerves have even gained 
access to the periphery and well before they 
have developed the capacity to transmit impulses. 



Therefore, in the first stages of life, responses 
to sensory stimulation are secondary, not pri- 
mary, for action. 

From the beginning, the embryo acts in an 
integrated fashion. Its first movements are mass 
actions. Later, more specialized and particular- 
ized actions arise through the individuation of 
motor patterns derived out of a background of 
mass actions. As development progresses, mass 
actions are gradually held in check until, in the 
mature organism, they provide mainly the tonal 
and postural adjustments stabilizing the plat- 
form upon which an individuated act is carried 
out. During the process of generating new pat- 
terns of activity, the nervous system continues to 
behave in an integrated fashion; newly inte- 
grated patterns are built up from older inte- 
grated patterns. The nervous system preserves 
a fundamental continuity of self-integrity during 
all adaptation. 

Nerves serving particular muscles seem to de- 
velop a unique specification, an endowment re- 
ceived according to the particular peripheral re- 
lations they establish. The specifications are 
different if deliberate transpositions are made in 
the peripheral motor organization. Something 
of the same sort occurs in the specification of sec- 
ond and higher order central sensory units, as we 
shall see later. These constitute the most ele- 
mentary phenomena of commitment. 

2. The origins of action. For each incoming 
or outgoing neuron there are at least five thou- 
sand central neurons whose activities are confined 
to the brain and spinal cord. The total number 
of these central neurons is estimated at a stag- 
gering ten billions units. The nervous system 
used to be thought of as being activated pri- 
marily through the influx of outside stimuli. 
However, when direct observation of individual 
neuronal units became possible, it was discovered 
that some cells in each sense organ maintain a 
degree of "steady state" activity even in the ab- 
sence of identifiable stimuli. Moreover, a sub- 
stantial proportion of neurons in the brain and 
spinal cord show "spontaneous" activity from 
early embryonic stages onward, even during 
sleep, hibernation and deep anesthesia. "Spon- 
taneous" activity and "steady state" activity are 
probably functionally equivalent; neither re- 
quires excitation from outside the neuron itself, 
although the rates of activity can be modified by 



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205 



outside influences. Spontaneously active units 
are found within all regions of the central nerv- 
ous system, even in aggregates of neurons sep- 
arated from the nervous system. Various gangli- 
onic masses throughout the brain interact with 
neighboring and even remote parts. The nervous 
system is thus made up of vast aggregates of 
neurons each of which exhibits its own activity, 
yet all of the aggregates are bound together into 
mutually interdependent transactional functions 
constituting an integrated whole. 

It is evident that the nervous system is ac- 
tive as well as reactive. It is built for action. 
Even the earliest sensory messages enter upon a 
performance-oriented transaction. In respect to 
sensory messages, the central neurons govern: 
(i) the degree to which any incoming signal can 
invade and alter the central transactional sys- 
tem, and (ii) the degree to which such influence 
may be reflected during any subsequent motor 
performance. It is obvious that no sensory sig- 
nal enters upon a tabula rasa. Neuronal mes- 
sages entering the central nervous system can 
achieve direct and indirect relations with a very 
large number of mutually interdependent aggre- 
gations of neurons already organized into an 
integrated system. 

3. Genetically determined and undetermined 
behavior. It is obvious that some sensory stimuli 
contribute quite directly to motor performance. 
Through genetically inscribed pathways, certain 
sensory signals can induce stereotyped reflex re- 
sponses. Nonetheless, even these imperious re- 
flex responses are clearly "conditioned" by on- 
going activity in nearby and even remote parts 
of the central nervous system. This feature sup- 
plies the principal diagnostic value to clinical 
reflex response testing. 

Through more elaborate but still genetically 
ordained paths, the sensory system can play a 
"releasing" and guiding role in what has been 
referred to as "instinctual" behavior. Although 
reflex behavior is relatively fixed and is not pro- 
foundly changed by maturation and learning, in- 
stinctual behavior may await certain stages of 
nervous system or endocrine maturation and may 
be subject to some modification according to ex- 
perience and learning. Beyond reflexive and in- 
stinctual systems, however, all of the rest of the 
central nervous system seems to be highly modi- 
fiable. Thus, reflex pathways are the least modi- 



fiable, most direct, and shortest-circuited; paths 
relating to instinctual behavior are longer-cir- 
cuited and more modifiable ; the remainder of the 
central nervous system organization is relatively 
long-circuited and uncommitted. 

Our problem relates chiefly to this long-cir- 
cuited, most modifiable compartment: What evi- 
dence is there that this system is not committed 
at birth? What evidence is there that this sys- 
tem can be committed to neuronal patterns relat- 
ing to experience and that, following such com- 
mitment, the system is relatively less free for 
re-commitment? 

It is reasonable to suppose that because of the 
vast numbers of neurons involved and because of 
the tenuousness of many of the mutually inter- 
dependent relations, extremely subtle changes in 
time and tide of activity in only a few units may 
have an enormous effect on the outcome of any 
given initial state. It is important to remember 
that such a system can only progress from one 
stage to the next, from an uncommitted, inte- 
grated performance to a committed but still in- 
tegrated performance. In each successive state, 
the organism is active and acting, even if that 
action is simply to hold more overt action in 
abeyance. The organism can never revert to its 
original uncommitted state but must instead pro- 
gress from one committed state to the next and 
so on. Presumably this progression will be re- 
tarded according to whatever in the next pattern 
of adaptation may be incongruous or in conflict 
with the presently ongoing integrated system. 

Pertinent to these organismic considerations is 
Whitehead's solution of the apparent conflict be- 
tween concepts of permanence and becoming, 
and between determinism and free will. The or- 
ganism becomes something else on the basis of 
its given ("permanent") endowment; direction of 
"free will" is based upon what has been deter- 
mined up to the present : direction is always from 
somewhere, from something. 

4. The processes of neuronal commitment. 

(a) Congenital absence of sense. Persons born 
totally blind never know what vision is like. They 
have no visual memories or visual dreams. They 
do not "see stars" or anything else "visual" if 
bumped on the head. Individuals who become 
blind after having some experience with sight, 
that is, after the first few years of childhood, 
usually retain for the rest of their lives powerful 



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memories of visual patterns. They may have 
vivid impressions of color and light elicitable by 
memory or dreaming. Kimio Eto, one of the 
best koto players in Japan, describes his visual 
experiences as follows: "I was very fortunate; 
when I went blind, I was old enough to know 
color quite vividly. As I play, certain notes and 
melodies bring back certain colors, in a perfect 
form. Somehow, I feel that my world must be 
one of the most beautiful." 

Persons with congenital cataracts who can see 
only diffused light during childhood do not have 
good vision immediately after the cataracts have 
been removed, even if they have satisfactory cor- 
rective lenses. It takes such individuals a long 
time to learn to employ visual signals inde- 
pendently of their other sense modalities in the 
identification of simple geometrical figures. It 
takes them even longer to develop any confidence 
of action in circumstances of visual dependence. 
It is doubtful whether they ever do see "nor- 
mally" if the cataract removal has been delayed 
beyond childhood. (A roughly parallel account 
is given of chimpanzees raised in complete dark- 
ness for some months. The animal studies con- 
trol for some of the ambiguities obtaining in pa- 
tients with congenital cataracts, e.g., that there 
might have been other congenital defects affect- 
ing vision, or interference by virtue of the sur- 
gery or post-operatively inadequate optical ac- 
commodation.) 

We are led by these facts to surmise: (i) in 
the absence of access to the outside world through 
a particular sensory modality, the otherwise ap- 
propriate brain pathways do not organize central 
representation of that aspect of the world, (ii) 
sensory experiences in early childhood may be 
lastingly committing with respect to central rep- 
resentation, and (iii) the absence of such child- 
hood experiences may be limiting with respect 
to the later fitness of that same system for cen- 
tral representation. 

(b) Phantom sensation. When a limb is con- 
genially missing or is amputated at birth or 
shortly thereafter, there is no evidence of phan- 
tom sensation referable to the missing part. 
Nevertheless, when a limb is amputated anytime 
after early childhood, the individual uniformly 
has a phantom which usually persists for the 
duration of his life. Phantoms may be associ- 
ated with the loss of fingers, toes, penis, breasts, 



ears or nose, but the most vivid and enduring 
phantoms follow major limb amputations. Con- 
scious awareness of phantom parts relate to those 
to which conscious attention is ordinarily di- 
rected ; thus, the phantom fingers, hand, and wrist 
may be vividly experienced, whereas the forearm 
and upper arm may be foreshortened and only 
vaguely experienced. The phantom can usually 
be described in detail as to its exact position in 
space, in relation to the rest of the subject's 
body, its motility, and its participation in "volun- 
tary" movements. There may be detailed sensa- 
tions relating to temperature sense, muscle ten- 
sion, joint position, nail bed, skin tension, tickle, 
whether hairs are erected or not, etc. Normally, 
phantoms can be caused to change position, to 
"move at the free will" of the subject. The sub- 
jective feeling of "thumbing your nose" with a 
phantom is said to be exactly equivalent to that 
of the normal extremity, just as simple but not 
as risky. 

We are led further to surmise: (i) the speci- 
fication of "sign" given to second and higher 
order sensory units by virtue of sensory experi- 
ence is integrated into patterns, (ii) these pat- 
terns involve sensory and motor integration, (iii) 
these central patterns continue to remain coor- 
dinated even in the absence of the peripheral 
organ (the limb) and the peripheral motor nerve 
endings and effectors and the sensory end-organs 
which are nominally considered to be responsible 
for the origination and organization of limb sen- 
sation. Peripherally assigned functions have 
been taken over, as it were, by second and higher 
order motor and sensory neurons which continue 
to function in accordance with their experienced 
commitments. This means that muscle, tendon 
and skin receptors activated in concert while ex- 
periencing active and passive movements of the 
previously intact limb have somehow conferred 
"pattern integrity" as well as "sign" upon higher 
order sensory and motor units relating to that 
limb. This "pattern integrity" and "sign" never 
appear without abundant explicit personal ex- 
periences. Yet commitment of childhood is ade- 
quate for an indefinite duration of the phantom, 
even in the absence of any further impulses from 
the missing extremity. Despite intellectual and 
visual evidence to the contrary, the phantom con- 
tinues to survive in conscious awareness. The 
previously experienced integrated patterns re- 






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207 



main inscribed and integrated according to pre- 
vious commitment. 

It might be interjected that the phantom rep- 
resents nothing more than "memory," but this 
begs the question: Where does this "memory" 
reside? The phantom (or "memory") can be 
modified by cooling the amputation stump, by in- 
jecting procaine or alcohol into the amputated 
nerve trunks and the local spinal sympathetic 
ganglia, and by spinal anesthesia. Furthermore, 
phantoms following transection of the spinal cord 
are specifically and characteristically different 
from phantoms following limb amputation. 
These facts combine to suggest that the phan- 
tom as a "memory" not only originates initially 
from ascending impulses but may, after the am- 
putation is performed, continue to have its 
"roots" in parts close to the original input stages. 

(c) Conditioning, learning, and the process of 
becoming. The natural history of developing 
perceptual, motor, cognitive and social skills is 
being studied in various branches of psychology, 
anthropology and sociology. Considerable is now 
known about conditioning and other forms of 
learning, including social imitation, and learn- 
ing dependent on the symbolic processes of lan- 
guage and mathematics. Learning in any form 
appears to depend upon: (i) confrontation of 
the subject by an unfamiliar configuration; (ii) 
devotion by the subject of attention to that con- 
figuration; (iii) existence in the subject of ap- 
propriate motivation or drive; (iv) development 
of a new central neuronal configuration yield- 
ing ideas or action relating to the unfamiliar con- 
figuration; and (v) feedback to the subject of 
information respecting success or failure, ap- 
propriateness or inappropriateness, of his ideas 
or actions. Failure of any one of these five es- 
sentials precludes learning anything at all. 

It is also obvious that the subject must make 
use of an internalized neuronal scheme of the 
universe in which he and his body parts are 
represented in organized ways which can be 
variously articulated with systems representing 
objects other than himself and with systems rep- 
resenting time and space. Very little of this 
arrangement appears to be genetically deter- 
mined, as we have observed. History teaches us 
that men have held very different notions about 
themselves and about objects around them. Cer- 
tainly, in the natural history of the individual, 



such a linked succession of different concepts of 
self and beyond self takes place. 

Early psychological studies emphasized the 
high degree of presumptive correspondence be- 
tween physical objects and the subjective report 
of these objects as perceived, and the high degree 
of correspondence among reports from different 
individuals observing the same object. This 
tended to stress the impartiality and consistency 
of the perceived world. Naturally, the first test 
objects were selected as being relatively unam- 
biguous. Naturally, the subjects were required to 
"pay attention" to the object and were given 
certain other stabilizing instructions. The sub- 
jects were advertently and inadvertently highly 
motivated to provide what might be considered 
"normal" subjective reactions. It was quickly 
recognized that "inattentiveness" or "fatigue" or 
"suggestion" have remarkable effects upon the 
perception. 

Certain objects turned out to be interpreted 
as having "size constancy" and other attributes 
which depend upon the subject making certain 
sophisticated assumptions concerning both space 
and object, both of which acts of assumption are 
based on previous and not on the immediate ex- 
periential conditions. Later it became fashion- 
able deliberately to influence perception by induc- 
ing in subjects certain prescribed expectations. 
Both past experiences and induced expectations 
are profoundly influential on percepts, judgments, 
and behavior. Still more recently, it was dem- 
onstrated that an individual may be converted in 
his perception or judgment of even simple and 
familiar geometrical configurations by confront- 
ing him with contrary ("incorrect") but con- 
certed views of other subjects. The interesting 
thing is that, under such conditions, objects come 
to appear to the subject as if they actually do 
correspond to the socially adopted view. Other 
investigators pointed out that children are more 
reliable witnesses than adults for certain kinds 
of objective reality. This is assumed to be be- 
cause children lack the persuasive commitments 
incorporated into the perceptual apparatus of the 
adult as a result of his longer experience. 

From infancy, through puberty, adulthood, 
"parentage" and finally old age, we are really a 
succession of different persons. We have dif- 
ferent appetites, perceptions, emotions, activities 
and different capacities for conscious awareness 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and responsible behavior. At different ages, we 
obviously have different schemes for ourselves 
and for the rest of the universe. Not only do 
we change rapidly, but the world changes rapidly 
around us. We tend to adjust ourselves to these 
changes through a combination of adapting our- 
selves to the circumstances and adapting circum- 
stances to our changing needs. In the process, 
we tend to underestimate the true extent of the 
alteration of our internal and external state. 
For instance, as adults, we would have a difficult 
time converting our body image back to a pre- 
puberty stage even though we have had abundant 
experience with that former personal body 
scheme. We have since that period become 
something else. We are continuously in the proc- 
ess of becoming. 

On a shorter time scale we are continually 
changing in ways that profoundly affect our per- 
ceptions, judgments and actions. For instance, 
when we are satiated, it is difficult for us to 
feel an adequate compassion and urgency for 
those who are hungry. For this reason, a good 
restaurant does not permit cooks and waiters to 
eat until after serving the customers. When we 
are food deprived for 24 hours or more, our at- 
tention is swiftly attracted and held by food 
odors, our judgment is biased respecting the 
value of, say, an onion, and our actions are 
both consciously and unconsciously dedicated to 
food procurement. Although such tides of ap- 
petite and satiety are generally short run, if a 
man is chronically starved or cold or penurious, 
he learns to take advantage of features of his 
environment not attended to by others and he 
may develop a life-long commitment relating to 
highly specialized perceptions, judgments and 
actions. 

The educational, social, political, military and 
economic changes which have revolutionized the 
pattern of lives within this generation are elo- 
quent testimony to change and to the range of 
human capabilities for adaptation to change. We 
can appreciate from the experience gained in 
these years the powerful influence of conscious 
and rational dedication to successful adaptation. 
We can likewise appreciate the stimulating as 
well as disruptive potentialities imposed by 
change. Yet, characteristically, we do not project 
into our views of the future a degree and rate 
of change equivalent to what we have gone 



through in the past. This limitation of pro- 
jective imagination tends to influence us to un- 
derestimate our powers for conscious dedication 
and rational election in the direction of success- 
ful accommodation among the various alterna- 
tives we are confronting. 

(d) Sensory deprivation. When normal sub- 
jects are placed in circumstances of sensory de- 
privation (really, a reduction or monotony of 
sensory input instead of true deprivation), they 
characteristically develop certain sensory, judg- 
mental and motor defects. This is true whether 
the deprivation is induced by confinement in a 
light-proof, sound-proof cubicle or submergence 
in a constant temperature pool of water. After 
some hours or at most 2 or 3 days of confinement, 
subjects begin to lose their normal ability to 
control their thought processes. They cannot 
sustain a line of thinking or bring to mind fa- 
miliar ideas. Soon thereafter hallucinations may 
appear, beginning with visualized geometrical 
patterns and going on to include auditory and 
somaesthetic impressions. Hallucinations there- 
after become more elaborate and intrusive, and 
eventually may involve elaborate perceptions of 
bodily movement, voices and moving scenes. The 
subject's ability to respond to his own or to out- 
side commands, to discriminate test objects, to 
think, and even to perform simple motor skills 
such as handwriting and walking, becomes dis- 
turbed. Recovery may take several hours or 
even days. 

One is left with an impression from such 
studies that contact with "reality" may be more 
dependent upon experience than we generally 
suppose. We presumably are normally encoun- 
tering frequent and automatic validations and 
cross-validations of our internal circuits through 
continually varying perceptual, judgmental and 
action experiences. This process may be con- 
tributing to the maintenance of "normalcy." 
When this process is substantially reduced as in 
sensory deprivation, "spontaneous" patterns of 
activity in the central nervous system may begin 
to emerge from controls depending upon con- 
tinuing perceptual, judgmental and behavioral 
experience. Such experiments have suggestive 
implications as to the nature of hallucinations 
experienced by lonely explorers, prisoners, truck 
drivers on long and monotonous runs, febrile and 






NATIONAL INSTITUTE OF MENTAL HEALTH 



209 



isolated patients, and perhaps also frank mental 
disorder. 

Pure-bred Scotty dog puppies have been sep- 
arated so that half of a litter could be raised in 
an experience-limiting environment and the other 
half in private homes. Over a period of months 
the experience-limited animals developed a per- 
sisting hyperactivity and an incapacity to learn 
simple discriminations and motor performances 
which were quickly comprehended by their sib- 
lings. The effects of upbringing in such cir- 
cumstances appear to be long lasting. 

We are led to conclude that the relationship 
between experience and the internal neuronal 
processes governing perception, ideation and ac- 
tion is one of great interdependence. Varied ex- 
perience may be indispensable in fostering and 
assuring continuity of a capacity to maintain 
contact with and to deal appropriately with a 
changing world. 

(e) Sensory stimulation. It is a commonplace 
experience to alight from long, noisy airplane 
flight and thereafter for some hours to experi- 
ence a continuing "noise in the head." Similarly, 
hair blowing on scalp or forearms for some 
hours may be followed by persisting sensations 
imitative of the "blowing hair" experience. Dis- 
embarking from a rough ocean crossing may be 
followed by continuing or recurrent sensations of 
motion of the now stable environment, as if it 
were a moving ship at sea. The percept may 
be associated with a wide-based, "rollicking" gait 
said to characterize the sailor ashore. The 
French refer to this illusion of motion as mal 
de debar quement. If the seas have become grad- 
ually rougher during the crossing, the first occa- 
sion for vomiting from sea sickness may be ex- 
perienced on solid land. 

One is led to interpret this as a phenomenon 
of temporary functional commitment. It under- 
lines the reality of ongoing central activity as a 
controlling force in perception and action, and 
illustrates once more an experience-imposed lack 
of correspondence between percept and outside 
reality. 

(f) Related internal processes. Recently de- 
veloped techniques have been introduced which 
make possible crude observations in experimental 
animals of internal changes accompanying adap- 
tive behavior. Only the most primitive and pre- 
liminary insights have been gained thus far, but 



the domain is vast and promising. A little is 
now known which can provide internal (neu- 
ronal) linkage patterns complementary to the 
external (behavioral) linkage patterns estab- 
lished by the social sciences. Obviously the 
nervous system provides whatever mechanisms we 
have not only for physiological coordination 
within ourselves but also for our comportment 
in the world around us including our manage- 
ment of social relations. 

It is now possible to delineate certain general- 
ized changes of functional activity within the 
brain which are associated with alterations be- 
tween sleep and wakefulness. The mechanisms 
of "arousal" and "attention" and the more par- 
ticularized "orienting reflex" are understood in 
a preliminary way. Activation of certain brain 
loci induces animals to behave as if they were 
being "rewarded"; activation of other regions 
has a "punishing" effect; activation of still other 
parts of the brain is neutral with respect to 
such reinforcements. Some parts of the brain 
are clearly associated with appetite and other 
parts, physiologically linked with these appeti- 
tive centers, are clearly related to emotion. Both 
the appetitive and emotion systems seem to be 
bound up in what we mean by the terms "motiva- 
tion" and "drive." A wide variety of condition- 
ing experiments has revealed some degree of 
modifiability of at least certain of these central 
circuits. Central responses to standard sensory 
signals, for example, are notably different de- 
pending on whether or not the test stimuli are 
associated with other "significant" (rewarding 
or punishing) stimuli. 

5. Effects of commitment. This section is 
based largely upon experiments conducted by the 
late Adelbert Ames, Jr. Although his work 
does not yet have a widespread influence in con- 
temporary thought, this is undoubtedly relateable 
to the difficulty imposed upon the communica- 
tion of insight into transactional mechanisms by 
means of languages built for the expression of 
linear cause and effect of relationships. Ames 
resorted principally to demonstrations to con- 
vey the meaning of his work. Ames's demon- 
strations, unfortunately, take quite as deliberate 
and conscientious study as do scientific papers. 
They are frequently mistakenly treated as if they 
were simple tricks and no more sophisticated than 
vulgar parlor magic. Nonetheless, the hypo- 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



theses and interpretations which were the reason 
for Ames's development of these demonstrations 
are among the most profound and far-reaching 
ideas concerning man's relations with the uni- 
verse including himself and his fellow man. John 
Dewey wrote that Ames's work "is by far the 
most important work done in the psychological- 
philosophical field during this century — I am 
tempted to say the only really important work." 

Ames's experiments deal with the perception 
of distance, objects, and motion, and with the 
sequential significances of objects in motion in 
relation to the observer and his capacity to an- 
ticipate the future course of such objects. He 
first demonstrates that the content of perceptual 
awareness includes striking contributions on the 
part of the observer. Such contributions are 
not obtainable from the objective reality of the 
present, but are based upon assumed familiari- 
ties which, in turn, depend upon the observer's 
experience with cues now recurrent in the pres- 
ent situation. Physiological events as well as 
object-originated cues are compounded in our 
perceptual awareness. Ames sought demonstra- 
tions of what we "subjectively" contribute to our 
perceptions of "something out there." Our past 
experiences contribute weighted information on 
what we have most usually experienced at times 
when we had similar sensory cues. Brightness, 
size, parallax, convergence and disparity are 
visual cues which contribute to assumptions about 
location and movement of objects. Character- 
istics such as hardness, malleability, resiliency, 
combustibility, etc. are assumed on the basis of 
more or less similar visual, auditory and somes- 
thetic cues. 

"We cannot reach an understanding of what 
the environmental situation does contribute to 
visual awareness if we think of the environment 
as an immediate, instantaneous existence inde- 
pedently apart from environmental situations 
earlier experienced by the observer or independ- 
ent or environmental situations we might experi- 
ence later." In encountering noncorrespondence 
between what we assume to be the reality of a 
given situation and our prediction of its conse- 
quences, we feel insecure as a function of the 
intensity of the discrepancy of cues. "Funda- 
mentally, our feelings of security or insecurity as 
to subjective significances arise from processes 



below the level of awareness * * * These sensed 
feelings may be the original value judgments." 
Ames's demonstrations show that we make in- 
stantaneous and complex adjustments to per- 
ceived cues in the direction of assuring corre- 
spondence between previous experiences and the 
present conscious awareness. For example, if 
you put on aniseikonic glasses and observe the 
room in which you are now sitting, you will in- 
stantaneously and automatically correct for the 
distortion imposed by the lenses in such a way 
as to maintain your percept of the room accord- 
ing to your previous experience with architec- 
tural symmetry. Through use of different lenses, 
different problems can be imposed upon your 
capacities to make the present percept conform 
to your previous experience. One pair of lenses, 
for example, can project on your retinae cues 
which correspond to the area of the ceiling be- 
ing twice that of the floor, the walls sloping 
out with appropriate changes in the angles re- 
lating walls to each other and to the floor and 
ceiling. Other lenses can be made to cause a 30° 
slope of the entire room. Nevertheless, almost 
all adults fail to see anything discrepant about 
their percept of the room which is given "nor- 
mal" room configurations. You may feel only 
mild unsureness about this complexly readjusted 
percept. After further experience with anise- 
ikonic glasses it is likely that you may become 
able to perceive the room according to the 
veridical visual cues actually being presented 
to your sense receptors, that is, as distorted. 
The basis for your previous instantaneous and 
automatic adjustment of the incoming cues to 
fit your past experience lies with you, the ob- 
server, and not with the cues. A beginning 
schoolchild, having less experiential commitment 
with the usual architectual symmetry of rooms, 
will far more quickly perceive the distortion. 
Presumably an adult savage would similarly 
more quickly perceive the room correctly, as 
distorted. 

If aniseikonic glasses which project an appar- 
ent slope to the environment are employed out- 
of-doors, the tilt may be recognized while looking 
at fields and rolling hills. But if the scene con- 
tains a lake, the tilt is automatically rejected 
in conscious awareness presumably because slop- 
ing water violates previous experience. If, in- 
stead of using aniseikonic lenses, a room is ac- 






NATIONAL INSTITUTE OF MENTAL HEALTH 



211 



tually distorted, you will perceive the room as 
normally symmetrical. But then individuals in 
the room are perceived as distorted in size ac- 
cording to experience dominated assumptions 
which you make about the symmetry of the room. 

Expectations as to future consequences ("se- 
quential significances") have their impact on ac- 
tions. If you are asked to bounce a rubber ball, 
a steel ball, or a golf ball, you will make ap- 
propriately different movements in order to catch 
the ball off the bounce. These movements are 
based upon previous experience with similar ob- 
jects. If a ball of putty is substituted, you will 
not expect it to bounce. But if the ball of putty 
is made of a silicone compound known as "silly 
putty," malleable although it is, it will bounce 
much more briskly than a rubber ball. In order 
to perceive the nature of objective reality and 
to predict its "sequential significance" and the 
influence of your actions on the succeeding se- 
quence of events, we automatically make a host 
of assumptions and predictions. The skill of an 
accomplished athlete depends on his ability to 
make accurately weighted assumptions on the 
basis of his extensive past experience, weighted 
particularly according to experiences most closely 
resembling the present. 

Demonstrations and experiments of this kind 
indicate that an observer develops conscious 
awareness only of those aspects of his surround- 
ings which "carry significances" to him. These 
significances sustain, extend, or vary the satis- 
faction of his purposes as these are carried out 
in action. "An observer's experiences have com- 
pounded into a 'subconscious' recognition that the 
significance of his environment is not disclosed 
by his perception of what and where 'objects' are 
but by the conseqquences of his purposeful be- 
havior guided by his prehensions that he knows 
his perceptions of objects are only symbolic ref- 
erents. * * * Learning occurs only in relation to 
events of which the learner has already had ex- 
perience and only under conditions of noncorre- 
spondence between his prehensions of sequential 
events and the sequences occurring." In other 
words, we admit to conscious awareness that 
which has meaning to us in relation to our pur- 
poses. We learn from those things admitted to 
conscious awareness which do not correspond to 
our basic assumptions. Overcoming a noncorre- 
spondence by inappropriate construction of past 



assumptions does not eventuate in learning new 
characteristics of the environment but in a lost 
opportunity for learning, and in a subsidence of 
the internally generated urge to establish corre- 
spondence. 

6. Conversion of commitments. About the turn 
of the century, G. M. Stratton, in a series of 
brilliant experiments, demonstrated that it is 
possible to induce rather complete conversions 
and reversals of committed patterns. Stratton's 
experiments have been widely replicated and 
extended since then but their mysteries are by 
no means plumbed. In brief, by means of lenses, 
it is possible to project on the retina an inver- 
sion of the visual field, or a reversal of left and 
right sides of the field, or a combination of both 
up-down and left-right reversals in the same pair 
of glasses. When you first wear such glasses 
there is considerable uneasiness and conflict, but 
after a matter of days of continuing experience, 
"correspondence" is restored ! An individual can 
thereafter ride a bicycle, fence, play golf and 
perform other visually dependent activities with 
ease and skill. 

In the process of getting used to an upside- 
down world, you begin by making elaborate con- 
scious thoughts concerning the course and im- 
provement of movements: you "know intellectu- 
ally" that it is necessary to move your hand 
downward in order to reach a nearby object 
which your conscious awareness labels as above 
your line of vision, but your previous commit- 
ments dictate otherwise. Making the movement 
in the right direction requires deliberation at 
first; many inappropriate actions take place. 
Later, entirely appropriate movements are made 
smoothly and automatically. Still later, and 
after a further period of experience, you discover 
that there is no further conflict of cues : the per- 
ceived world is restored to its traditional "right- 
side-upness." Learning to adjust to a left-right 
reversal is more difficult, requiring severalfold 
longer experience. Learning to adjust to the 
combined up-down and left-right reversals is yet 
more difficult. 

In these experiments all identifications of the 
outside world attaching to "thereness" have to 
be specifically relearned in all visual patterns 
despite the non-correspondence of this require- 
ment with past visual, somesthetic and motor 
experiences. When the glasses are removed a 



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ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



similar (but distinctly shorter) period of re- 
adjustment to the normal configuration is re- 
quired. That is, soon after the glasses are re- 
moved, you cannot perform simple acts like rid- 
ing a bicycle because of the new "noncorre- 
spondences" between incoming cues and the as- 
sumptions built up during experiences obtaining 
while wearing the reversing lenses. The right- 
side-up world briefly appears "upside-down" 
once more. 

The general principle involved here has been 
extended with roughly analogous results in rela- 
tion to motion and color as well as form cues in 
vision, auditory localization reversals, transplan- 
tation of tendons and muscles, ect. Apparently 
man can accommodate certain kinds of conver- 
sion of long standing and elaborately inscribed 
commitments. This is not possible for the sala- 
mander, however. Visual field reversals in the 
salamander are inappropriately responded to in 
an enduring fashion. Presumably, somewhere 
between lower and higher vertebrates a certain 
degree of freedom for functional reorganization 
has been acquired. 

7. Interpretive formulations relating to these 
observations. In this section we intend being 
deliberately speculative. To some extent we are 
discussing "a bag of cats and dogs." Never- 
theless, the following tentative suppositions com- 
mend themselves for consideration: 

(a) Not much of the nervous system is in- 
dubitably and inevitably committed in the be- 
ginning, not even the motor cells of the "final 
common path." The motor and sensory units 
become most firmly committed by virtue of the 
particular attachments they assume. The second 
and higher order units associated with them are 
less imperiously committed. Even circuits de- 
voted to instinctual behavior are subject to some 
influence through experience. Neuronal mecha- 
nisms still more remote from direct input and 
output relations may be still more modifiable by 
experience. 

(b) The commitment of motor and sensory 
units seems to be quite firmly established early in 
life. The commitment of second and higher 
order sensory and motor units appears to take 
place on the basis of the "sign" committed by 
particular muscle and sensory end-organs and 
also on the basis of the "pattern integrity" built 
up through coordinated sensory and motor ex- 



periences. Body image formation apparently 
occurs early in life and may last indefinitely. 

(c) Other kinds of commitment take place in 
a more plastic, alterable form. The neuronal 
circuitry of this latter variety is still mostly a 
mystery and calls for further analysis. Appar- 
ently it is an endowment of higher evolutionary 
forms. Somehow experiences in perception, judg- 
ment and action are laid down in unconsciously 
operating patterns which contribute in a con- 
tinuing way to conscious awareness and behavior. 
Past experiences are weighted according to the 
familiarity of apparent recurrences in the pres- 
ent and are closely bound up with one's aims and 
purposes. It is impossible to disentangle which 
parts of any ongoing perceptual experience are 
based on "objective reality" and how much is 
contributed from an unconscious synthesis of our 
present sensory cues, our previous experiences 
and our purposes. As Ames has established, we 
cannot be aware of the relative proportions of 
the working triad: outside object, conceptual 
content, and purposes, excepting through pains- 
taking experimental isolation of the three inter- 
dependent components. This is the main busi- 
ness of science. But the pragmatic solution of 
"what works" and the universality of experi- 
ence, which are so useful in most other areas of 
science and technology, are difficult to apply to 
human relations because of the requirement to 
obtain a full consciousness and consideration of 
the attendant assumptions which in turn relate 
to idiosyncratic individual and cultural experi- 
ences. The homogeneity of the "stuff" of con- 
templation of the physical sciences is wanting 
here. 

(d) Internal processes of commitment occlude 
from our perception those things which are lack- 
ing in significance for us. This makes it difficult 
to perceive changes in our environment excepting 
in accordance with the extent of whatever fa- 
miliarities attach to an otherwise novel situa- 
tion; novel aspects of the environment need to 
be painstakingly apprehended, through familiar- 
izing experience with the non-correspondences ob- 
taining between our assumptions and forecasts on 
the one hand and what later happens. Learning 
depends entirely on our facing up to the non- 
correspondences between our environment and 
our upbringing to that moment. Failure to 
reckon with noncorrespondences precludes our 



NATIONAL INSTITUTE OF MENTAL HEALTH 



213 



learning anything or achieving diversified growth 
or departing from the dead center of our present 
limited existence. Failure to deal with noncor- 
respondences limits our grasp of nature and re- 
duces our predictive capabilities upon which 
survival and well-being depend. 

(e) Internal processes tend automatically to 
dispose of noncorrespondences through a process 
of internal misprehension of the non-familiar. 
It is as though the nervous system, having to 
work upon learning, lazily precludes acceptance 
of non-correspondences for as long as it can — 
that is, until failure of behavioral achievements, 
assignable to failure to find a mechanism for cor- 
respondence, enlarges our purposes to correct 
that defect. Misprehension results in an instan- 
taneous and comprehensive oversight or sys- 
tematic misinterpretation of all of the incon- 
gruent, noncorresponding cues. This failure is 
likely to occur whenever the incoming incon- 
gruent cues are weak or are dominated by fa- 
miliar corresponding cues. This is especially 
likely to occur in a social context among persons 
having essentially the same background of ex- 
periences. There is a powerful tendency toward 
conformity in perceptions, judgments and ac- 
tions resulting from the socially integrative pur- 
poses of being considered "normal" and of con- 
sidering oneself a "normal." This tendency for 
social conformity powerfully reinforces our in- 
ternal experience-endowed assumptions and fore- 
casts. 

(f) It is not only difficult to perceive non- 
correspondences, but learning is entirely depend- 
ent upon our recognition of such noncorrespond- 
ence (consciously or unconsciously) within a 
framework of a purposive desire for survival 
and growth. 

(g) It is evident that we are not only con- 
tinually weighting assumptions according to our 
experiences and purposes but evidently we de- 
pend upon a continuing normalizing influence of 
sensory messages in order to maintain functional 
control over the relations between outside stimuli 
and our conscious awareness. Thus, sensory de- 
privation is followed by a relaxation of cor- 
respondence between "objective reality" and con- 
scious awareness and, also, by a relaxation of the 
internal controls by which we make use of our 
conscious awareness. 

(h) To continue the process of becoming, to 



increase our comprehension of the characteristics 
of ourselves and the physical and social universe 
around us, to learn, to create and to achieve any- 
thing new requires: (i) exposure to conditions 
leading to a noncorrespondence between our pro- 
jections based on past experiences, purpose and 
evolving events; (ii) recognition by us of the 
non-correspondence (this implies that significance 
is attached to the non-correspondence with re- 
spect to our purposes) ; (iii) devotion of atten- 
tion to those characteristics which specify the 
non-correspondence (this usually involves making 
predictions and producing ideas and actions 
which may be more or less appropriate) ; (iv) 
development in us of a new body of weighted 
assumptions now competent to correspond to the 
formerly non-correspondent features of the sit- 
uation; and (v) feed-back of information re- 
specting the appropriateness and inappropriate- 
ness of our newly adjusted predictions and 
actions. These requirements include specific in- 
sights fundamental to learning. 

(i) The nervous system is built for action. 
This action is always expressive of a dynamic 
flux of purposes generated within the organism. 
The nervous system is integrated in the function 
of its parts and as a whole. Recurrent patterns 
of stimuli impinging from the outside may be 
represented and re-represented in central pat- 
terns, but always in conservative relationship to 
previously established ongoing patterns and in 
conformity to presently obtaining purposes. 

Implications Relating to National Government 
and Diplomacy 

Here, we intend to consider national sur- 
vivability, adaptability and growth in the light 
of implications arising from our consideration 
of individual existences. The first and most ob- 
vious recommendation contributed by the anal- 
ogy is the need to establish and identify inte- 
grated national purposes. The purposes of a 
nation, like those of an individual, are manifold 
and always in flux. Some are short-term and 
others long-range. National purposes demand 
purposeful dedication and responsibility among 
the nation's citizens and allies, public identifica- 
tion of individual and group accomplishments 
in the light of national purposes, and some means 
for the expression of national satisfaction in the 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



development and exercise of national capabilities. 
A wise nation, like a wise individual, has worthy 
goals, denned in terms of long-range potentiali- 
ties. The ultimate achievement of any nation 
depends upon the creation of institutions which 
embody national ideals and upon the degree of 
aspiration and self-discipline exercised by its 
servants and its citizens. The pursuit of na- 
tional excellence, like the pursuit of individual 
excellence, is an ennobling and liberating as well 
as disciplining enterprise. 

A nation, like a man wishing to be understood, 
identifies and displays its purposes so as to aid 
others to understand and to anticipate appro- 
priately its national actions. Narrow and nega- 
tive purposes, and purposes inadequately pro- 
jected, present handicaps to communication. This 
is especially important to bear in mind because 
purposes affect foreign as well as national per- 
ceptions, judgments and actions. National pur- 
poses expressed for export should be entirely 
congruent with those conveyed internally. As 
with an individual, the flux of national purposes 
contains conflicts. This may call for alternations 
of effort in support of conflicting short-term pur- 
poses and to keep the country moving in that 
general direction. 

It would seem appropriate in a nation, as in an 
individual, to benefit by directing attention to 
noncorrespondences between national assumptions 
and predictions based on past experience and the 
actual sequence of events. As with an individual, 
discovery of such noncorrespondences is difficult. 
First, it is frustrated by the assumption that 
the behavior of other nations and national repre- 
sentatives will be consistent with our limited im- 
pression of their world-view, experience and pur- 
poses. Second, the discovery of noncorrespond- 
ences is frustrated by any national influence in 
the direction of national conformity of views. 
The goal in attending to noncorrespondences is 
to become as fully aware as possible of all char- 
acteristics of objective reality. We need not be 
blind, nationally: but this requires a positive 
policy of cultivating persons able to perceive, 
and encouraged to perceive, noncorrespondences 
between our national percepts, ideas and actions 
and the objective events of history. There is a 
positive national need for persons who under- 
stand the previous experiences and purposes of 
other nations so thoroughly that they are able 



to make better predictions than those based on 
our own national sets of assumptions and pur- 
poses. It takes positive national purpose to be 
receptive and responsive to evidences of noncor- 
respondence. 

A third requirement is for abundant and 
spirited activity on a national scale; activity 
which acknowledges the inevitability of change; 
activity which recognizes the processes of be- 
coming on a national scale. Our nation should 
have the zest to utilize the potentialities offered 
by change rather than to suffer passively from 
the limitations imposed by change. National a.c- 
tivity should correspond with the evolution and 
achievement of national purposes, to the percep- 
tion of "national self" and the "national self" 
in relation to the rest of the world. National 
activity should reflect the evolution and adjust- 
ment of national perspectives, concepts and 
ideals. National activity should be balanced by 
rational insight into the fact that it is impos- 
sible to succeed one hundred percent of the 
time, that it may be more deadly to be afraid 
of making mistakes than to make them. There 
needs to be a national willingness to act tenta- 
tively, to utilize what turns out to be inappro- 
priate action as a key to growth, to be internally 
and externally candid about misdirected and in- 
correct interpretations and actions and, on occa- 
sion, to withhold action in favor of a theoretical 
testing out of alternatives in the search for a more 
desirable course of action. Such a trial of alter ^ 
natives can yield insight into more dimensions 
than can be discovered simply by the traditional 
techniques of predication. 

We need a national scheme of working as- 
sumptions based upon objectively evaluated past 
experiences projected realistically into the future, 
weighted according to present and projected 
needs of the nation. This scheme should be 
open-ended, plastic and modifiable. It should 
be as objectively oriented to reality as possible 
through a continuing testing against events as 
they take place. Such a scheme will reveal many 
non-correspondences but should not itself be con- 
sidered infallible. The chief difficulty lies in 
keeping a scheme of working assumptions suffi- 
ciently dynamic and future-oriented. The only 
safeguard resides in having institutional mecha- 
nisms for objectifying information concerning 
outside relations. This is improved by maximiz- 



NATIONAL INSTITUTE OF MENTAL HEALTH 



215 



ing international transactions. I suppose that a 
nation can suffer from isolation from the rest of 
the world in ways analogous to an individual 
exposed to sensory deprivation. In such cir- 
cumstances, we might expect patterns based on 
past experience to interfere with incoming sig- 
nals and with the objectification of national self- 
awareness. 

Science can play a valuable role in relation to 
national security and welfare according to this 
scheme. First, it is the professional business 
of science to discover noncorrespondences. Sci- 
ence advances on the basis of the discovery of 
discrepancies between present limited concepts 
and the behavior of nature. The techniques of 
science are directed toward the objectification of 
these discoveries and their interpretation and 
application in the largest and most general sense. 

Second, science characteristically builds models 
and makes internal trial-and-error exercises. 
Sometimes these are entirely mental processes; 
they may involve elaborate equipment. By 
means of experiments, scientists attempt to grasp 
the significant relations of natural phenomena 
without having to replicate nature in its entirety. 
Science can play an important role in the testing 
of alternative perceptions, judgments and activi- 
ties and in the selection of techniques prior to 
the undertaking of expensive and committing 
national actions. 

Third, science continually practices the devel- 
opment of schemes of weighted assumptions 
which are as far as possible generalized to in- 
clude all sense-experiences, both past and future. 
These are open-ended schemes of science, always 
accessible to new evidence of non-correspondence. 

Fourth, as a system of thought, science de- 
mands a high degree of intellectual freedom, is 
not imposed by coercion or persuasion, and is 
not destroyed when found internally inconsistent. 
Paradoxically, science becomes stronger and more 
co